A medicament for use in a method of inducing or extending a cellular cytotoxic immune response

ABSTRACT

The present invention relates to a medicament for use in a method of inducing a cellular cytotoxic immune response, the method comprising the steps of: i) administering to a patient a delivery system comprising (a) a molecule binding to a receptor on the surface of a dendritic cell, (b) an antigen-comprising protein bound to molecule of (a) and (c) a first adjuvant, wherein upon binding of the molecule of (a) to the receptor, the protein of (b) is internalized and processed in the dendritic cell and the antigen comprised in the protein is presented on the surface of the dendritic cell, thereby activating a T cell in the patient; and ii) administering to the patient a re-activator selected from the group consisting of (d) complexed interleukin 2 (IL-2cx), (e) a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, and (f) a combination of (d) and (e), wherein the peptide is derived from the antigen-comprising protein as defined in step i), thereby reactivating the T cell activated in step i), wherein the re-activator of step ii) is administered in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

The present invention relates to a medicament for use in a method ofinducing a cellular cytotoxic immune response, the method comprising thesteps of:

-   -   i) administering to a patient a delivery system comprising (a) a        molecule binding to a receptor on the surface of a dendritic        cell, (b) an antigen-comprising protein bound to molecule of (a)        and (c) a first adjuvant, wherein upon binding of the molecule        of (a) to the receptor, the protein of (b) is internalized and        processed in the dendritic cell and the antigen comprised in the        protein is presented on the surface of the dendritic cell,        thereby activating a T cell in the patient; and    -   ii) administering to the patient a re-activator selected from        the group consisting of (d) complexed interleukin 2        (IL-2cx), (e) a peptide-loaded major histocompatibility complex        class I (MHC-I) presenting cell and a second adjuvant, and (f) a        combination of (d) and (e), wherein the peptide is derived from        the antigen-comprising protein as defined in step i), thereby        re-activating the T cell activated in step i),        wherein the re-activator of step ii) is administered in a time        frame of from 0 h to 14 days after the administration of the        delivery system of step i).

The immune system protects the body against pathogens and tumor cells bya variety of mechanisms. To function properly, it has to discriminatebetween “self” and “foreign” (pathogens/tumors). It detects and fights avariety of pathogens, including bacteria, viruses, parasites, fungi, andtoxins. The immune systems of vertebrates such as humans consist of manytypes of proteins, cells, tissues, and organs, which interact in adynamic network. As part of this complex immune response, the vertebrateimmune system adapts over time to recognize particular pathogens moreefficiently. The adaptation process creates immunological memory andallows a more effective protection during future encounters with thesepathogens. Vaccination is based on this process of acquired immunity.

Dendritic cells (DCs) form part of the immune system. Their mainfunction is to process antigen material and present it on their surfaceto other cells of the immune system, thus functioning asantigen-presenting cells.

T helper cells (also known as effector T cells or T_(h) cells) are alsoan important member of the immune system in that they play a fundamentalrole in establishing and maximizing the capabilities of the immunesystem. T_(h) cells are involved in activating and directing otherimmune cells. They are essential in determining B cell antibody classswitching, in the activation and expansion of cytotoxic T cells, and inmaximizing bactericidal activity of phagocytes such as macrophages. Itis this diversity in function and their role in influencing other cellsthat gives T helper cells their name. Proliferating helper T cells thatdevelop into effector T cells differentiate into two major subtypes ofcells known as T_(h)1 and T_(h)2 cells (also known as Type 1 and Type 2helper T cells, respectively), wherein T_(h)2 cells mainly promote thehumoral immune system (stimulation of B cells into proliferation,induction of B cell antibody class switching, and increase of antibodyproduction), whereas T_(h)1 cells promote mainly the cellular immunesystem (maximization of killing efficacy of the macrophages andcytotoxic CD8⁺ T cells). Depending on the nature of the invadingpathogen, the immune system develops a Th1 or Th2 immune response. Inthe case of the Th1 immune response, the CD8⁺ T cells show a strongtendency for differentiation into cytotoxic T cells. At the same time,both the CD8⁺ and CD4⁺ helper T cells of the Th1 immune response secretelarge amounts of IFN-γ (and other Th1 cytokines/chemokines) and elicitthe generation of antibodies predominantly of the IgG2a and IgG2bisotype in the mouse and predominantly of the IgG isotype in the human.The Th1 immune response is particularly effective for defending the bodyagainst viruses, (intracellular) bacteria and tumors.

Currently available vaccines and adjuvant systems directed against live,attenuated, or inactivated pathogenic components mainly elicit anantibody immune response, but not an effective Th1 cytotoxic response(Steinman et al., 2007, Nature 449, 419-26). The induced antibodies bindto components of the pathogen and thus biologically inactivate it(“neutralizing antibodies”). However, there are a number of diseases,where neutralizing antibodies are not sufficient to protect from thedisease or to control the disease and current vaccine technology is noteffective. These are diseases which may require an effective Th1 immuneresponse for containment and/or eradication of the infection. Examplesare tuberculosis, malaria, leishmania, prion diseases, orthomyxovirusesand in particular influenza, hepatitis A, hepatitis B, humanimmunodeficiency virus (HIV) and other lentiviruses, cytomegalovirus,herpesviruses, papillomaviruses, bunyaviruses, caliciviruses,filoviruses, flaviviruses and in particular hepatitis C virus,papillomaviruses, paramyxoviruses, a variety of respiratory viruses, andother viruses which need for containment and eradication an effectiveTh1 immune response, and in particular a Th1 cytotoxic response. Thedevelopment of a vaccination methodology inducing such an effective Th1response is therefore highly desirable. Furthermore, targeting themechanism of “cross-presentation” (see below) is of paramount importancefor the induction of a Th1 immune response against viral, bacterial,parasitic, and fungal pathogens, since dendritic cells most often do notbecome directly infected in the course of an infection. Without thedevelopment of a Th1 immune response, many viral, bacterial, parasitic,or fungal infections cannot be contained or eradicated in the humanbody.

It has been found in WO 2009/065561, that cells playing a major role inthe induction of the Th1 response can be selectively targeted. It wasfound that chemokine (C motif) receptor 1 (XCR1) is present on thesurface of professional antigen-presenting cell, particularly dendriticcells (DC), and can be used to selectively deliver substance into thesecells. Targeted delivery of a substance to XCR1-bearing DC allowsinduction of a potent Th1 immune reaction in mammals/humans. Currentvaccines mainly address the Th2 antigen presentation pathway and mainlylead to the generation of Th2-type (neutralizing) antibodies and immunereactions. In particular, through targeting to XCR1-bearing DC, aTh1-type humoral and cellular (cytotoxic) immune reaction can beelicited to a given immunogen. It can be anticipated that NK cells, CD8⁺T cells, and Th1CD4⁺ T cells participate in this reaction, but otherCD4⁺ T cells may also contribute to this type of reaction. An adjuvant,either alone or in combination with an immunogen or any pharmaceuticalcompound, can be selectively targeted to XCR1-bearing antigen-presentingcells (APC).

In the periphery, the immune system has to discriminate between harmlessforeign or self-antigens on the one hand and dangerous (viral,bacterial, fungal, parasitic, toxin-like) antigens on the other hand.The antigen is taken up by the DC and broken down to peptides(“processed”). The resultant peptides are “presented” to T lymphocytes(T cells) in the context of the MHC class I or MHC class II. The CD4⁺subset of T cells recognizes the antigen in the context of MHC class II,the CD8⁺ subset of T cells recognizes the antigen in the context of MHCclass I. Concomitant with the uptake of antigen, the DC is capable ofsensing through a large set of “danger signal” recognition receptors(e.g. toll-like receptors, NOD-like receptors), whether the antigen isof dangerous nature or whether it is harmless. The patterns recognizedby the “danger signal” recognition receptors (also designated “patternrecognition receptors”) are usually molecular structures that are uniqueto microorganisms. These can be cell wall components (e.g.lipopolysaccharide, peptidoglycan) or nucleic acid modifications (e.g.unmethylated CpG motifs) in case of microbes, or structural features andmodifications that are unique to viral DNA or viral RNA (e.g.double-stranded RNA). Also cells dying from apoptosis in the bodyrelease molecules which are capable of triggering “danger signal”recognition receptors (e.g. High Mobility Group Protein B1, heat-shockproteins).

In the case of a dangerous antigen, the DC activates a specific responseprogram (“maturation”). The antigen is presented to CD4⁺ and CD8⁺ Tcells, which simultaneously receive from the DC additional signalsindicating the dangerous nature of the antigen. As a result, both T cellsubsets become activated, expand extensively with a prolonged life spanand develop to “effector T cells”. These can be CD4⁺ T cells providing“help” to other DC or B cells or other cells of the immune system, orcan be even CD4⁺ cytotoxic cells. Within the CD8⁺ T cell subset, again Thelper cells develop, but a large proportion of CD8⁺ T cells becomeeffector cells capable of eliminating the invading pathogen throughsecretion of IFN-γ and other soluble factors or through killing ofinfected body cells. As a result of the T cell help to B cells,antigen-specific B cells differentiate to plasma cells which secreteantibodies directed to the antigen (pathogen). These antibodies help tofight the pathogen through a number of mechanisms (e.g. neutralization,improved antigen uptake, opsonization, complement fixation).

A certain number of effector CD4⁺ and CD8⁺ T cells survive the acutephase of an immune response to a pathogen and become long-lived “memoryT cells”. Memory T cells and long-lived plasma cells orchestrate uponre-exposure to the same pathogen (antigen) a very fast immune responseallowing the immune system to eliminate the pathogen (antigen) veryeffectively. This enhanced capability of the T-cell and B cell immuneresponse upon re-exposure to the same pathogen is termed “immunity” andthe antigens which induce immunity are “immunogenic”.

In summary, the T cell compartment of the immune system contains CD4⁺ Tcells and CD8⁺ T cells. In the naïve organism, only a few hundred CD4⁺or CD8⁺ T cells recognize a given antigen. As long as they have notencountered antigen, T cells are in a naïve state and cannot exerteffector functions. Naïve CD4⁺ and CD8⁺ T cells can only be activated byAPC by soluble protein or polypeptide antigen, which is taken up by theAPC, processed, and “presented” on the cell surface in the context ofMHC. The source of primary antigen exposure can be a soluble protein orpolypeptide or any type of vaccine, such as attenuated infectiousagents, viral or bacterial vectors coding for a desired antigenicprotein or peptide, or DNA or RNA expression vectors coding for anantigenic protein or peptide. As a result of primary exposure to antigenin the context of danger signals there will exist within days apopulation of “primed” CD4⁺ and CD8⁺ T cells.

When protein antigen is targeted into an APC, it will be degraded(“processed”) to peptides and these peptides will be presented on thesurface of the APC in the context of MHC-II (classical presentation) andMHC-I (“cross-presentation”). Naïve CD4⁺ T cells recognize theirpeptide-antigen in the context of MHC-II, naïve CD8⁺ T cells in thecontext of MHC-I. They become activated, proliferate, and acquireeffector functions. If the peptides are presented by the DC in thecontext of a “danger signal”, the expanded CD8⁺ T cells willdifferentiate into cytotoxic T cells. Only professional APC are capableof inducing this cytotoxic function in naïve CD8⁺ T cells (“priming”,“primary immunization”). Once the CD8⁺ T cells have acquired theircytotoxic potential, they will be able to kill cells in the body whichexpress the same peptides in the context of MHC-I. Thus they will killcells infected by a given infectious agent or tumor cells.

Antigens which are taken up by cells capable of antigen presentation arenot only presented in the context of MHC-I (“cross-presentation”), butalso in the context of MHC-II (“classical presentation”). Therefore, anyantigen delivery into antigen-presenting cells, together with a dangersignal, will not only activate CD8⁺ T cells, but also CD4⁺ T cells. TheCD4⁺ T cells will differentiate into Th1 T cells secreting TNF-α andIFN-γ, and some will also develop into cytotoxic T cells.

Primary CD8⁺ T cell activation can be achieved by providing a solubleantigen, or by direct or indirect targeting of antigen(s) into APC (Bcells, macrophages, DC), and in particular into XCR1⁺ DC of a naïvehost. Indirect targeting is achieved by providing the antigen in cellswhich are not APC, but which can hand over the antigen to APC, inparticular to XCR1⁺ DC in vivo. When the antigen is applied togetherwith a “danger signal” (e.g. LPS, Poly I:C, CpG, etc.), this will induceprimary CD8⁺ T cell cytotoxic immunity.

Similar early T cell activation can be expected by antigenicre-challenge (in the presence of danger signals) of a host, which hasbeen primed with antigen before and whose antigen-specific CD8⁺ T cellsand CD4⁺ T cells are in a non-activated memory state.

Similar T cell activation can be expected in hosts which harbor lowgrade chronic infections which cannot be cleared by the host (e.g. CMV,HCV, HIV).

Antigen can be targeted into APC. This targeting can be mediated bymonoclonal antibodies (mAb) or antibody fragments binding to surfacestructures on APC, or by any ligands binding to receptors on the surfaceof APC. These ligands can be sugar moieties, chemokines, soluble proteinligands, or any other structures allowing internalization of the antigeninto the APC. Antigen taken up by any DC is presented both in thecontext of MHC-I and MHC-II. Direct or indirect targeting of antigeninto dendritic cells (DC) substantially improves cross-presentation overother modes of antigen application. Best cross-presentation and thuspriming of CD8⁺ T cells can be achieved by targeting of antigen intoXCR1⁺ DC (Bachem et al. 2010, J Exp Med 207, 1273-1281, Bachem et al.,2012, Front Immunol 3, 214; Caminschi et al. 2013, Radford et al. 2013,Kreutz et al. 2013).

After an efficient priming of CD8⁺ T cells in the context of a dangersignal (which is also a first adjuvant), these cytotoxic T cells willrepresent approximately 1% to 5% of the CD8⁺ T cell repertoire andexhibit significant cytotoxic potential. For example, the achievedcytotoxicity may protect the host against a pathogen, from which theantigen was derived, and this level of protection can also be effectivefor newly developing cancerous tissue (FIG. 2B).

However, when attempting to establish long-term immunity against a givenantigen or to provide high levels of cytotoxic protection againstimminent infections, or to provide high levels of cytotoxicity againstalready established tumors, the cytotoxic potential induced by aninitial priming of naïve CD8⁺ T cells or be re-activation of CD8⁺ Tcells may not suffice. Under these circumstances a further amplificationof the activated antigen-specific CD8⁺ T cells is necessary.

After recognizing antigen expressed in the context of MHC-I, CD8⁺ Tcells become activated, de novo express or strongly upregulate a varietyof cell surface molecules, such as CD69, 4-1 BB, ICOS, CD25, CD40L,OX40, and proliferate for up to approximately 8 days in the mouse (thetimeframe may be somewhat different in the human). Thereafter, theinitially activated CD8⁺ T cells gradually return over approximately 2-3weeks into a resting “memory” state. At the same time, the expandedantigen-specific T cell population strongly contracts. A proportion ofthe CD8⁺ T cells will survive this process of contraction and willbecome memory T cells (Cui et al., 2010, Immunol. Rev. 236, 151-166).

Classical vaccination schemes can be divided into an initial primingstep, followed by one or several “boostings”. The principle of boostingis based on de novo activation of initially expanded B cells or T cellswhich have undergone a downmodulation of the initial activation or havealready reverted into the resting state.

The problem underlying the present invention is the provision of animproved medicament for use in a method for inducing a cellularcytotoxic immune response and/or in a method for extending a cellularcytotoxic immune response compared to prior art technologies.

The problem is solved by the present invention.

In one embodiment, the present invention relates to a medicament for usein a method of inducing a cellular cytotoxic immune response, the methodcomprising the steps of:

-   -   i) administering to a patient a delivery system comprising (a) a        molecule binding to a receptor on the surface of a dendritic        cell, (b) an antigen-comprising protein bound to molecule of (a)        and (c) a first adjuvant, wherein upon binding of the molecule        of (a) to the receptor, the protein of (b) is internalized and        processed in the dendritic cell and the antigen comprised in the        protein is presented on the surface of the dendritic cell,        thereby activating a T cell in the patient; and    -   ii) administering to the patient a re-activator selected from        the group consisting of (d) complexed interleukin 2        (IL-2cx), (e) a peptide-loaded major histocompatibility complex        class I (MHC-I) presenting cell and a second adjuvant, and (f) a        combination of (d) and (e), wherein the peptide is derived from        the antigen-comprising protein as defined in step i), thereby        re-activating the T cell activated in step i),        wherein the re-activator of step ii) is administered in a time        frame of from 0 h to 14 days after the administration of the        delivery system of step i).

In a preferred embodiment, the medicament is for use in a human.

The patient is preferably a mammalian patient, more preferably a humanpatient. The patient may be suffering from an infection or tumor, or, incase of prophylactic treatment, is not suffering from an infection ortumor, but is to be protected from a respective infection or tumordisease.

In a further preferred embodiment, the receptor on the surface of adendritic cell is a human receptor on the surface of a dendritic cell.

One possibility to provide strong T cell cytotoxicity within a shorttime would be to interfere with the down regulatory mechanism becomingeffective within days of the initial activation or re-activation of CD8⁺T cells by antigen. To prevent this down modulation of the CD8⁺ T cellresponse, we reasoned that we have to provide another stimulus involvingthe T cell receptor complex on CD8 T cells and/or appropriate growthfactors within a short timeframe of the initial activation orre-activation. This concept runs against the current dogma which teachesthat repeated activation of T cells via the T cell receptor within ashort time will result in “activation-induced cell death”(Gorak-Stolinska et al., 2001, J Leukoc Biol 70, 756-766).

To test our concept, C57BL/6 mice were immunized (“primed”) by targetingan antigen into XCR1⁺ DC in the presence of a first adjuvant whichsupports a Th-1 response (poly I:C, LPS, CpG, or equivalent). As antigenfor this primary immunization we used the model antigen ovalbumin (OVA),or a peptide sequence derived from ovalbumin (SIINFEKL (SEQ ID NO: 11)).This peptide sequence is known to be preferentially presented in thecontext of the MHC-I of C57BL/6 mice after processing of OVA by thetargeted APC. For this initial immunization, the protein OVA or thepeptide SIINFEKL (SEQ ID NO: 11) were recombinantly fused either to amonoclonal antibody (mAb) specific for XCR1 or to the chemokine ligandXCL1 binding to XCR1, as described earlier (WO 200/065561). At varioustime points (3-20 days) after this priming step, mice were re-exposed toantigen. We did not inject protein or peptides into the host, since weanticipated that either procedure would lead to the presentation ofantigenic peptides in the context of the MHC-I on host cells, which thenwould be killed by the already activated antigen-specific CD8⁺ T cells(compare FIG. 1A), a highly undesired effect. Instead, syngeneic spleniccells were isolated from C57BL/6 mice, incubated with the peptideSIINFEKL (SEQ ID NO: 11) in vitro (“loading” of MHC-I), washed, andinjected i.v. into mice which have been primed before.

Unexpectedly, and contrary to current knowledge, we could achieve withthis regime an amplification of the number of antigen-specific CD8⁺ Tcells, when the CD8⁺ T cells were re-exposed to antigen within a narrowtimeframe. No amplification was observed, when re-exposure was veryearly, on day 3, and very limited amplification was observed whenre-exposure to antigen was done following day 9 of initial CD8⁺ T cellactivation (FIG. 3). Injection of SIINFEKL-loaded syngeneic spleniccells into primed animals alone did not expand the primed CD8⁺ T cellpopulation, only when a second adjuvant (poly I:C, LPS, CpG, orequivalent) was co-applied to provide a “danger” signal, the desiredeffect was achieved. When the injection of peptide-loaded splenocyteswas performed at an optimal time point, the antigen-responsive CD8⁺ Tcell population expanded approximately 10-fold, from 0.2×10⁶ afterpriming without amplification, to 2×10⁶ cells with amplification (FIG.4). These expanded CD8⁺ cells expressed high levels of effectormolecules such as Granzyme B, perforin, TNF-α and IFN-γ, moleculesinvolved in the CD8⁺ T cell defense of infectious agents or in theeradication of tumors. In the human, the optimal timeframe for theamplification of the initial T cell activation may differ from theoptimal timeframe in the mouse (approximately day 5-8 after initial CD8⁺T cell activation).

When comparing various amplification time points, it became apparentthat a re-exposure with antigen 5-8 days after the initial priming givesthe highest degree of CD8⁺ T cell expansion and the highest expressionof cytotoxic effector molecules (TNF-α, IFN-γ, granzyme B, perforin) inCD8⁺ T cells. Day 5-8 is an early time point following the recognitionof antigen by resting CD8⁺ T cells and thus a time point at which the Tcells are still strongly activated. Therefore, this amplification doesnot represent a classical boost system. Instead, this type ofamplification provides signals allowing T cells to continue theirinitial activation and expansion phase instead of entering the usualphase of downregulation and contraction. Because of this particulareffect, we have termed this amplification, when applied together with anadjuvant, as “Antigen-Dependent Amplification System” (ADAS).

Therefore, according to the present invention, “Antigen-DependentAmplification System” or “ADAS” is understood as the second step ii) ofthe present invention relating to administering to the patient are-activator, wherein the reactivator is a peptide-loaded majorhistocompatibility complex class I (MHC-I) presenting cell and a secondadjuvant, and wherein the peptide is derived from the antigen-comprisingprotein as defined in step i) of the present invention, therebyre-activating the T cell activated in step i). Optionally, thereactivator in ADAS further comprises complexed interleukin 2 (IL-2cx).

In order to examine the cellular requirements for the effectiveness ofADAS, various lymphocytic populations (splenocytes, B cells, T cells,and DC) were for comparison loaded with SIINFEKL (SEQ ID NO: 11) invitro and injected within the optimal timeframe in mice (day 5) togetherwith a second adjuvant (poly I:C, LPS, CpG, or equivalent). Thisexperiment determined that all of these lymphocytic populationsexpressing MHC-I were capable of providing the signals necessary tocontinue the initial activation, expansion and functionaldifferentiation of CD8⁺ T cells to cytotoxic effector cells (FIG. 3B).While priming alone resulted on day 10 in 1%-5% of antigen-specific CD8⁺T cells within the splenic CD8 T cell population, application ADASraised this frequency to around 15%-25%.

From the results obtained one can conclude that ADAS will work in vivowith any system capable to provide high enough density of peptide-loadedMHC-I molecules on the surface of lymphocytic cells or evennon-lymphocytic cells. Instead of loading MHC-I molecules with peptidesexternally in vitro, one could envisage systems in which cells would befed in vitro with whole antigen-comprising protein, allowing the cellsto process the antigen and present the antigenic peptides in the contextof MHC-I. One could also envisage systems, in which cells would be invitro exposed to viral systems capable of infecting the cells resultingin the expression of high amounts of a peptide in the context of MHC-I.Further, the MHC-I bearing cells could be transfected with expressionvectors coding for a given protein or peptide sequence, again resultingin an efficient presentation of peptides in the context of MHC-I.

Since whole antigen delivered to APC will not only be presented in thecontext of MHC-I to CD8⁺ T cells, but also in the context of MHC-II toCD4⁺ T cells, ADAS can also be used to amplify CD4⁺ T cell responses.For this particular amplification, the cells used for ADAS have toexpress MHC-II molecules on the cell surface, which would be loaded withappropriate peptides. This loading could be done by external exposure tosuitable peptides, or the MHC-II bearing cells could be transfected byexpression vectors coding for a given protein or peptide sequence, againresulting in an efficient presentation of peptides in the context ofMHC-II.

Although we applied ADAS through i.v. injection, other routes ofapplication of the peptide-loaded cells are possible. This could be doneby subcutaneous, intracutaneous, intramuscular injection,intraperitoneal, intrathecal, or by direct injection into tumor tissue.

Therefore, the administration of a reactivator of step ii) of thepresent invention may be performed by known methods of administration,in particular selected from subcutaneous (s.c.), intracutaneous, i.v.,intramuscular injection, intraperitoneal, intrathecal, or by directinjection into tumor tissue, more preferably by i.v. or s.c. injection.

Also, the administration of a delivery system of step i) of the presentinvention may be performed by known methods of administration, inparticular selected from subcutaneous, intracutaneous, i.v.,intramuscular injection, intraperitoneal, intrathecal, administrationinto the lung, or by direct injection into tumor tissue, more preferablyby i.v. and s.c. injection.

The molecule binding to a receptor on the surface of a dendritic cell,(b) an antigen-comprising protein bound to molecule of (a) and (c) afirst adjuvant are preferably administered as a single pharmaceuticalpreparation. Such pharmaceutical preparation is preferably a liquidwhich may further contain pharmaceutical acceptable excipients likebuffering compounds. Such pharmaceutical preparation is preferablysterilized.

The volume of the dose of the delivery system of step i) forintramuscular administration is preferably up to about 5 mL, forexample, between 0.3 mL and 3 mL, between 1 mL and 3 mL, about 0.5 to 1mL, or about 2 mL. The amount of active ingredient in each dose shouldbe enough to provide for treatment or prevention. In differentembodiments, the unit dose of substance to be delivered should be up toabout 5 μg substance/kg body weight, between about 0.2 to 3 μg, betweenabout 0.3 to 1.5 μg, between about 0.4 to 0.8 μg, or about 0.6 μg. Inalternative embodiments unit doses could be up to about 6 μgsubstance/kg body weight, between about 0.05 to 5 μg, or between about0.1 to 4 μg. Representative amounts of protein per dose are fromapproximately 1 μg to approximately 1 mg, more preferably fromapproximately 5 μg to approximately 500 μg, still more preferably fromapproximately 10 μg to approximately 250 μg and most preferably fromapproximately 25 μg to approximately 100 μg.

The number of cells of a reactivator comprising a peptide-loaded majorhistocompatibility complex class I (MHC-I) presenting cell and a secondadjuvant to be administered in step ii) can vary and is typically in therange of from 1×10⁶ to 400×10⁶ cells, preferably is in the range of from1×10⁶ to 200×10⁶ cells.

Complexed IL-2 as reactivator is preferably administered as a solutionand/or at a dosage in the range of from 1 μg/kg body weight to 200 μg/kgbody weight, more preferably of from 1 μg/kg body weight to 50 μg/kgbody weight, even more preferably of from 1 μg/kg body weight to 20μg/kg body weight wherein the amounts refer to the IL-2 content in thecomposition.

Although we tested the effect of ADAS after targeting of antigen intoXCR1⁺ DC, one can conclude from our results that any system leading to asignificant initial activation (“priming”) or re-activation of CD8⁺ Tcells and/or CD4⁺ T cells by T cell receptor triggering in vivo can beamplified with the ADAS approach.

The lymphokine IL-2 has been used in the past in order to expand T celland NK cell populations and was shown to be effective, when provided ina “complexed” from, i.e. bound to an antibody blocking its binding tothe high affinity IL-2 receptor chain (CD25, Boyman et al., 2006,Science 311, 1924-1927).

According the present invention, “complexed IL-2” or “IL-2cx” isunderstood as IL-2 which is non-covalently bound to a binding molecule,in particular antibody or antibody fragment which is blocking itsbinding to the high affinity IL-2 receptor chain (CD25). In a preferredembodiment, the antibody is humanized or human. In a preferredembodiment, IL-2 is human IL-2. IL-2 may be synthesized synthetically orrecombinantly, using an adequate host, more preferably IL-2 is preparedrecombinantly.

We tested complexed IL-2 (IL-2cx) in our system of T cell activationthrough targeting of antigen into XCR1⁺ DC. Contrary to expectations,priming of the T cell response followed by application of IL-2cx alonedid not significantly increase the number of antigen-specific CD8⁺ Tcells (FIG. 4). Application of ADAS alone on day 5 increased the numberof antigen-specific CD8⁺ T cells from around 0.2×10⁶ after priming aloneto around 2×10⁶ and thus approximately 10-fold, as described above.However, and very surprisingly, when applied in the context of ADAS,IL-2cx very strongly augmented the amplification of antigen-specificCD8⁺ T cells to approximately 100-200×10⁶ antigen-specific CD8⁺, i.e.approximately by a factor of 50-100. This highly synergistic effectindicated that ADAS created favorable conditions for the biologicaleffects of IL-2, when IL-2 was applied in a complexed form. Thecombination of ADAS and IL-2cx was amplifying the initial priming tosuch a degree that now the large majority of all splenic immune cellswere composed of antigen-specific CD8⁺ T cells. When further examined,this massively expanded T cell population expressed granzyme B toapproximately 60-70% indicating a high cytotoxic potential.

In order to test the cytotoxic capacity of CD8⁺ T cells amplified undervarious conditions we chose a tumor model, in which a highly aggressiveOVA-transfected tumor line is injected s.c. into syngeneic C57BL/6 miceand allowed to grow for 6 days to a substantial size (around 20 mm²).From day 6, groups of mice were either left untreated, or were treatedby different regimes. Priming of the tumor-bearing mice on day 6 aloneby targeting of OVA into XCR1⁺ DC barely had any effect on the growth ofthe tumor (FIG. 5) and the same was true if the mice were not primed,but treated with IL-2cx alone. Surprisingly, priming of the mice on day6, followed by a treatment with IL-2cx alone for the consecutive dayssignificantly reduced the growth of the tumor until day 22, indicatinginduction of substantial killing capacity in vivo. However, the tumorresumed its growth around day 22 indicating that not all of the tumormass could be removed. Also the application of ADAS on day 6 afterpriming very substantially reduced the growth of the tumor which,however, re-started to grow around day 18 (FIG. 5). Interestingly, theapplication of ADAS on day 6 after priming combined with consecutiveapplication of IL-2cx was clearly most effective in reducing the size ofthe tumor until the end of the experiment (FIG. 5). The obtained resultsindicated that injection of IL-2cx alone within a given timeframe aftertargeting of antigen into APC was already very effective in controllingthe growth of the tumor. Further, the outcome indicated that ADAS, whenapplied within a given timeframe after initial activation of CD8⁺ Tcells by antigen, also induced strong killing activity against a tumorexpressing this antigen. Most effective in controlling the growth of anaggressive tumor was a combination of ADAS and IL-2cx (FIG. 5). Althoughin this experiment ADAS was applied first and followed by IL-2cx, thetreatment sequence could also be reversed. In this case IL-2cx would beapplied first, followed at an appropriate time by ADAS.

In preferred embodiment of step i) of the medicament for use in a methodof the present invention is disclosed in WO 2009/065561 for XCR1 asreceptor on the surface of a dendritic cell. As disclosed in WO2009/065561, an anti-XCR1 antibody or fragment thereof, or XCL1 or afunctionally active fragment thereof can preferably be employed asmolecule binding to a receptor on the surface of a dendritic cell. Thedisclosure of WO 2009/065561 relating to the delivery system whereinXCR1 is receptor on the surface of a dendritic cell is herebyincorporated by reference and the embodiments disclosed therein alsoapply for step i) of the medicaments for use of the present invention.

Suitable antigen-comprising proteins are known to the skilled person.

For example, numerous suitable antigen-comprising proteins in thecontext of tumor diseases are known. Moreover, suitable peptides asvaccines are described, as e.g. summarized in Aranda et al.,Oncolmmunology 2:12, e26621; December 2013 for solid neoplasms,including glioma, lung carcinoma, sarcoma, melanoma, esophageal squamouscell carcinoma, gastric cancer, hepatocellular carcinoma, pancreaticcancer, colorectal carcinoma, renal cell carcinoma, prostate cancer,ovarian carcinoma, gynecologic malignancies, and various other tumors.The antigen-comprising proteins specifically targeted in these clinicaltrials encompass cancer-testis antigens such as NY-ESO-1, TTK proteinkinase (also known as MOS), lymphocyte antigen 6 complex, locus K (LY6K,best known as URLC10), insulin-like growth factor 2 mRNA binding protein3 (IGF2BP3, best known as IMP3), ring finger protein (RNF43), andtranslocase of outer mitochondrial membrane 34 (TOMM34);carcinoembryonic antigens like glypican-3; differentiation antigens suchas melan-A (MLANA) and premelanosome protein (PMEL, best known asgp100); tumor-restricted antigens, such as the SYT-SSX fusion (which isselectively expressed by synovial sarcomas as a result of at(X;18)(p11;q11) chromosomal translocation); as well as so-called“shared tumor-associated antigens” (antigens that are overexpressed bymalignant cells but also produced in normal amounts by one or severalhealthy tissues), including vascular endothelial growth factor receptor1 (VEGFR1) and VEGFR2, survivin, Wilms tumor 1 (WT1), telomerase reversetranscriptase (TERT), and p53. Such proteins are therefore suitableantigen-comprising proteins in the context of tumor. A further summaryon peptide vaccines in cancer is Yamada et al., Cancer Sci, 2013,104(1):15-21.

In case of inducing a cellular cytotoxic immune response in a tumorpatient or a patient to be prophylactically treated regarding a tumor, asuitable antigen-comprised protein involved in tumor disease may beused. Such proteins are known in the art. As described e.g. in Tackenand Figdor (Tacken P. J., Figdor C. G.; Targeted antigen delivery andactivation of dendritic cells in vivo: Steps towards cost effectivevaccines. Semin. Immunol., 2011, 23(1):12-20), ideal tumor antigens arethose known as shared tumor-specific antigens because they areselectively expressed in tumor cells, of various histotypes, and not inMHC-expressing normal tissues. Examples of such type of antigens are theMAGE-A or NY-ESO-1 antigens. The various members in this category ofantigens are expressed at variable proportions depending on the tumortype and the disease stage. Thus, vaccines based on these antigensrequire selection of patients bearing tumors that express the targetantigen. Another category of tumor antigens that are deemed valuable forvaccine development are those derived from oncogenic proteins which areoverexpressed in tumors. Bona fide non-self tumor antigens are derivedfrom two major sources: viral antigens in the case of tumors ofoncogenic viral origin such as cervical carcinomas caused by HPVinfection, and somatic mutations.

In case of inducing a cellular cytotoxic immune response in patientsuffering from an infection or a patient to be prophylactically treatedregarding an infectious disease (or infection), suitableantigen-comprised proteins of a pathogen may be used, in particular apathogen selected from malaria, tuberculosis, leishmania or a virus, inparticular a virus selected from an orthomyxovirus, influenza virus,hepatitis A virus, hepatitis B virus, chronic hepatitis C virus, alentivirus, in particular HI-Virus, cytomegalovirus, a herpes virus, apapillomavirus, a bunyavirus, a calicivirus, a filovirus, a flavivirus,or a respiratory virus. In an even more preferred embodiment,antigen-comprised protein is a protein of a virus selected from ahepatitis C virus, a papillomavirus, a paramyxovirus, or a respiratoryvirus.

Viruses, in particular RNA viruses, typically exhibit a high mutationrate. However, there are typically conserved regions found in particularin genomic segments encoding non-structural and/or internal proteins,such regions encoding a viral polymerase or a nucleoprotein. Suchregions are unsuitable for classical vaccination technologies, as suchconserved proteins are not exposed on the viral coat surface. Incontrast, such antigen-comprised proteins and/or antigen comprised inthe protein may be used in the medicament for use according to theinvention.

In a preferred embodiment, the antigen-comprised protein and/or antigencomprised in the protein of a virus is conserved.

In a further preferred embodiment, the antigen-comprised protein is anon-structural protein and/or the antigen is comprised in anon-structural and/or internal protein.

In a yet further preferred embodiment, the antigen-comprised protein isa protein of an RNA virus.

A “non-structural” or “internal” protein is a protein which is not partof the coat or envelope of a virus particle.

In a further embodiment, the antigen is comprised in the protein in stepi) is immunodominant. Immunodominant means that, although many pMHCcomplexes are available for a certain pathogen, T-cell responses arereproducibly focused on one or few key antigens, and such the antigen isone such key antigens.

Suitable antigen-comprised proteins and antigens of influenza A virusare for example described in Wu et al. (PNAS, 2011, 108(22): 9178-9183),and encompass NP (nucleoprotein), basic polymerase 1 and Ml.

Step ii) relates to administering to the patient a re-activator selectedfrom the group consisting of (d) complexed interleukin 2 (IL-2cx), (e) apeptide-loaded major histocompatibility complex class I (MHC-I)presenting cell and a second adjuvant, and (f) a combination of (d) and(e), wherein the peptide is derived from the antigen-comprising proteinas defined in step i), thereby re-activating the T cell activated instep i).

In an embodiment, in case of (e), the cell and the second adjuvant areadministered in a time frame of from 0 h to 14 days after theadministration of the delivery system of step i).

In one preferred embodiment, in case of (e), the cell and the secondadjuvant are administered only once in a time frame of from 0 h to 14days after the administration of the delivery system of step i).

In a preferred embodiment, in case of (e), the cell and the secondadjuvant are administered in a time frame of from 48 h to 14 days afterthe administration of the delivery system of step i).

For example, the cell and the second adjuvant are administered 48 h, 72h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11days, 12 days, 13 days or 14 days after the administration of thedelivery system of step i).

In a more preferred embodiment, in case of (e), the cell and the secondadjuvant are administered in a time frame of from 3 days to 9 days, evenmore preferably of from 4 days to 8 days, for example 4 days, 5 days, 6days, 7 days, 8 days after the administration of the delivery system ofstep i).

In another preferred embodiment, in case of (e), the cell and the secondadjuvant are administered only once in a time frame of from 0 h to 14days, preferably in a time frame of from 48 h to 14 days after theadministration of the delivery system of step i).

For example, the cell and the second adjuvant are administered only once48 h, 72 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days or 14 days after the administration ofthe delivery system of step i).

In a further preferred embodiment, in case of (e), the cell and thesecond adjuvant are administered only once in a time frame of from 3days to 9 days, even more preferably of from 4 days to 8 days, forexample 4 days, 5 days, 6 days, 7 days, 8 days after the administrationof the delivery system of step i).

In case of (d), complexed interleukin 2 (IL-2cx) is administered in atime frame of from 0 h to 14 days after the administration of thedelivery system of step i).

In case of (d), complexed interleukin 2 (IL-2cx) is preferablyadministered repeatedly after the administration of the delivery systemof step i) in a time frame of from 0 h to 14 days after theadministration of the delivery system of step i). It is furtherpossible, that further administration of complexed IL-2 takes placeafter 14 days after the administration of the delivery system of stepi); however, at least one administration is in a time frame of from 0 hto 14 days.

In a further preferred embodiment, in case of (d), complexed interleukin2 (IL-2cx) is preferably administered in a time frame of from 48 h to 9days, even more preferably of from 3 days to 8 days, for example 3 days,4 days, 5 days, 6 days, 7 days, 8 days after the administration of thedelivery system of step i).

In a yet further preferred embodiment, in case of (d), complexedinterleukin 2 (IL-2cx) is preferably administered repeatedly in a timeframe of from 48 h to 9 days, even more preferably of from 3 days to 8days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days afterthe administration of the delivery system of step i). Thus, complexedinterleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 times in a time frame of from 48 h to 9 days, even morepreferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days,6 days, 7 days, 8 days after the administration of the delivery systemof step i). For example, complexed interleukin 2 (IL-2cx) may beadministered daily or every two days. For example, complexed interleukin2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7days and 8 days after the administration of the delivery system of stepi). In another example, complexed interleukin 2 (IL-2cx) may beadministered 48 h, 4 days, 6 days, and 8 days after the administrationof the delivery system of step i).

It is further possible, that further administration of complexed IL-2takes place after the indicated time of, e.g. 14 days, preferably 9 or 8days, after the administration of the delivery system of step i);however, at least one administration is in the indicated time frame.

In case of (f), a combination of complexed interleukin 2 (IL-2cx), and apeptide-loaded major histocompatibility complex class I (MHC-I)presenting cell and a second adjuvant is administered. In a preferredembodiment, the complexed interleukin 2 (IL-2cx) may be administeredbefore, concomitantly or after administration of the peptide-loadedmajor histocompatibility complex class I (MHC-I) presenting cell and asecond adjuvant. Preferably, the peptide-loaded major histocompatibilitycomplex class I (MHC-I) presenting cell and a second adjuvant are onlyadministered once, and the complexed interleukin 2 (IL-2cx) isadministered repeatedly in a time frame of from 0 h to 14 days after theadministration of the delivery system of step i). In such embodiment,complexed IL-2 may be administered both before, concomitantly and/orafter administration of the peptide-loaded major histocompatibilitycomplex class I (MHC-I) presenting cell and a second adjuvant.

For the alternative (f), the same preferred embodiments foradministration of complexed IL-2 and peptide-loaded majorhistocompatibility complex class I (MHC-I) presenting cell and a secondadjuvant, respectively, apply as for alternatives (e) and (f).

In one embodiment, in case of (f), the cell and the second adjuvant areadministered only once in a time frame of from 0 h to 14 days after theadministration of the delivery system of step i).

In a preferred embodiment, in case of (f), the cell and the secondadjuvant are administered in a time frame of from 48 h to 14 days afterthe administration of the delivery system of step i).

For example, in case of (f), the cell and the second adjuvant areadministered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days or 14 days after theadministration of the delivery system of step i).

In a more preferred embodiment, in case of (f), the cell and the secondadjuvant are administered in a time frame of from 3 days to 9 days, evenmore preferably of from 4 days to 8 days, for example 4 days, 5 days, 6days, 7 days, 8 days after the administration of the delivery system ofstep i).

In another preferred embodiment, in case of (f), the cell and the secondadjuvant are administered only once in a time frame of from 0 h to 14days after the administration of the delivery system of step i).

For example, the cell and the second adjuvant are administered only once48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days or 14 days after the administration of thedelivery system of step i).

In a further preferred embodiment, in case of (f), the cell and thesecond adjuvant are administered only once in a time frame of from 3days to 9 days, even more preferably of from 4 days to 8 days, forexample 4 days, 5 days, 6 days, 7 days, 8 days after the administrationof the delivery system of step i).

In case of (f), complexed interleukin 2 (IL-2cx) is preferablyadministered repeatedly after the administration of the delivery systemof step i) in a time frame of from 0 h to 14 days after theadministration of the delivery system of step i). It is furtherpossible, that further administration of complexed IL-2 takes placeafter 14 days after the administration of the delivery system of stepi); however, at least one administration is in a time frame of from 0 hto 14 days.

In a further preferred embodiment, in case of (f), complexed interleukin2 (IL-2cx) is preferably administered in a time frame of from 48 h to 9days, even more preferably of from 3 days to 8 days, for example 3 days,4 days, 5 days, 6 days, 7 days, 8 days after the administration of thedelivery system of step i).

In a yet further preferred embodiment, in case of (f), complexedinterleukin 2 (IL-2cx) is preferably administered repeatedly in a timeframe of from 48 h to 9 days, even more preferably of from 3 days to 8days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days afterthe administration of the delivery system of step i). Thus, complexedinterleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 times in a time frame of from 48 h to 9 days, even morepreferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days,6 days, 7 days, 8 days after the administration of the delivery systemof step i). For example, complexed interleukin 2 (IL-2cx) may beadministered daily or every two days. For example, complexed interleukin2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7days and 8 days after the administration of the delivery system of stepi). In another example, complexed interleukin 2 (IL-2cx) may beadministered 48 h, 4 days, 6 days, and 8 days after the administrationof the delivery system of step i).

It is further possible, that further administration of complexed IL-2takes place after the indicated time of, e.g. 14 days, preferably 9 or 8days, after the administration of the delivery system of step i);however, at least one administration is in the indicated time frame.

Therefore, in a further preferred embodiment, in case of (f),

-   (A) the cell and the second adjuvant are administered in a time    frame of from 0 h to 14 days, preferably 48 h to 14 days, more    preferably in a time frame of from 3 days to 9 days, even more    preferably of from 4 days to 8 days, for example 4 days, 5 days, 6    days, 7 days, 8 days after the administration of the delivery system    of step i), even more preferably only once in a time frame of from    48 h to 14 days, more particularly only once in a time frame of from    3 days to 9 days, even more particularly only once in a time frame    of from 4 days to 8 days, for example 4 days, 5 days, 6 days, 7    days, 8 days after the administration of the delivery system of step    i), and-   (B) complexed interleukin 2 (IL-2cx) is administered in a time frame    of from 0 h to 14 days, more preferably 48 h to 9 days, even more    preferably of from 3 days to 8 days, for example 3 days, 4 days, 5    days, 6 days, 7 days, 8 days after the administration of the    delivery system of step i), most preferably administered repeatedly    in a time frame of from 0 h to 14 days, more preferably 48 h to 9    days, even more preferably of from 3 days to 8 days, for example 3    days, 4 days, 5 days, 6 days, 7 days, 8 days after the    administration of the delivery system of step i).

It is further possible, that further administration of complexed IL-2takes place after 14 days after the administration of the deliverysystem of step i); however, at least one administration is in a timeframe of from 0 h to 14 days.

In a yet further preferred embodiment, in case of (f), complexedinterleukin 2 (IL-2cx) is preferably administered repeatedly in a timeframe of from 48 h to 9 days, even more preferably of from 3 days to 8days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days afterthe administration of the delivery system of step i). Thus, complexedinterleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 times in a time frame of from 48 h to 9 days, even morepreferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days,6 days, 7 days, 8 days after the administration of the delivery systemof step i). For example, complexed interleukin 2 (IL-2cx) may beadministered daily or every two days. For example, complexed interleukin2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7days and 8 days after the administration of the delivery system of stepi). In another example, complexed interleukin 2 (IL-2cx) may beadministered 48 h, 4 days, 6 days, and 8 days after the administrationof the delivery system of step i).

It is further possible, that further administration of complexed IL-2takes place after the indicated time of, e.g. 14 days, preferably 9 or 8days, after the administration of the delivery system of step i);however, at least one administration is in the indicated time frame.

In another preferred embodiment, in case of (f), the cell and the secondadjuvant are administered only once in a time frame of from 0 h to 14days after the administration of the delivery system of step i).

The peptide-loaded major histocompatibility complex class I (MHC-I)presenting cell and the second adjuvant are preferably administered as asingle pharmaceutical preparation. Such pharmaceutical preparation ispreferably a liquid which may further contain pharmaceutical acceptableexcipients like buffering compounds. Such pharmaceutical preparation ispreferably sterilized.

In a preferred embodiment of the medicament for use,

-   (x) the complexed interleukin 2 (IL-2cx) is administered repeatedly,    in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times,    even more preferably wherein the complexed interleukin 2 (IL-2cx) is    administered every 1 or 2 days, and/or is administered repeatedly    during 5 days to 1 month, even more preferably 1 to 2 weeks, and/or-   (xx) the peptide derived from the antigen-comprising protein has a    length of 8, 9 or 10 amino acids and/or is a peptide presented by a    MHC-I, preferably by allele HLA-A2, HLA-A1, HLA-A3, HLA-B7, HLA-B35,    HLA-A24, or HLA-A30, more preferably by allele HLA-A2.

In a preferred embodiment, the complexed interleukin 2 (IL-2cx) isadministered repeatedly, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 times, even more preferably wherein the complexedinterleukin 2 (IL-2cx) is administered every 1 or 2 days, and/or isadministered repeatedly during 5 days to 1 month. Therefore, it ispossible, as described above, that further administration(s) ofcomplexed IL-2 take place after the timeframe of 14 days, or 9 or 8days.

As described above, a peptide-loaded major histocompatibility complexclass I (MHC I) presenting cell and a second adjuvant are preferablyadministered as reactivator in step ii) of the medicaments for use ofthe present invention. Moreover, the peptide is derived from theantigen-comprising protein as defined in step i). “the peptide isderived from the antigen-comprising protein” is understood as that thepeptide sequence is part of the antigen-comprising protein sequence(i.e. is a subsequence of antigen-comprising protein sequence).

A “peptide-loaded major histocompatibility complex class I (MHC I)presenting cell” is understood as cell which presents desired peptideson the surface of the cells by binding to MHC-I.

In one embodiment, the loading can be achieved by in vitro loading ofMHC I molecules with peptides externally as described in the examples.Alternatively cells can be fed in vitro with whole antigen-comprisingprotein, allowing the cells to process the antigen and present theantigenic peptides in the context of MHC I, or they are exposed in vitroto viral systems capable of infecting the cells resulting in theexpression of high amounts of a peptide in the context of MHC I, or theMHC I bearing cells could be transfected with expression vectors codingfor a given protein or peptide sequence, again resulting in an efficientpresentation of peptides in the context of MHC I.

Methods for determining a peptide for the loading of cells in thecontext of MHC-I are known in the art. For example, the methodsdescribed in Wu et al. (PNAS, 2011, 108(22): 9178-9183) may be used.

In case of applying defined peptides for loading, e.g. by externallyloading cells in vitro, the peptide sequence may be chosen by methodsknown in the art. Externally loading cells in vitro can be performed bymethods known in the art, e.g. by providing an aqueous solution of thepeptides, adding the solution to the cells, which are preferably in abuffered solution or medium, incubating the cells with the peptides asto achieve a high saturation of the MHC-I and/or MHC-II with therespective peptide, and optionally washing the cells, e.g. with anaqueous solution.

In one preferred embodiment, cells are loaded in vitro with one peptidewhich has a sequence which is a subsequence of the antigen-comprisingprotein. For example, an aqueous solution comprising one such peptidemay be added in vitro to a cell population, which is preferably a cellpopulation of the patient.

Alternatively, a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentpeptide loaded-cell major histocompatibility complex class I (MHC I)presenting cell populations may be used in step ii), wherein the cellsare loaded with different peptides, and wherein the cells are preferablycells of the patient.

Such mixture of cells may be obtained by incubating a cell population,like PBMC cells, with a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent peptides, thereby obtaining cells loaded with differentpeptides in the context of MHC-I. Alternatively, separate cellpopulations, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cellpopulations, may be incubated in vitro with different peptides, forexample 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides. Thereby,separate major histocompatibility complex class I (MHC I) presentingcell populations each loaded with a different peptide are therebyobtained. The different peptide loaded-cell major histocompatibilitycomplex class I (MHC I) presenting cell populations may be administeredseparately in step ii), or a mixture of the different peptideloaded-cell major histocompatibility complex class I (MHC I) presentingcell populations may be prepared, which may then be administered in stepii).

In case different peptides are employed, in particular if 2, 3, 4, 5, 6,7, 8, 9, 10 or more different peptides are employed, such peptides maybe derived from the same or a different antigen-comprising protein. In apreferred embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentpeptides derived from one tumor antigen may be used. This means, thatthe sequence of each peptide is a subsequence of the tumor antigen. Thesequences of such different peptides may be overlapping ornon-overlapping. In a further embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore different peptides derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent tumor antigen may be used. In such embodiment, the differenttumor antigens are related to the same or different tumor, preferably tothe same tumor.

Preferably, a peptide sequence of a length of 8, 9 or 10 amino acids ischosen, as peptides presented by MHC-I are typically of this length.

As further described in Wu et al., HLA alleles are extremelypolymorphic. Therefore, the peptide loaded is preferably a peptidepresented by a frequent HLA alleles. Therefore, in humans, a peptidepresented the most frequent allele HLA-A2 is particularly preferred.Alternatively, peptides presented by HLA-A1, -A3, -B7, -B35, which arethe alleles relevant for individuals of Caucasian origin may be used.HLA-A24 may be used for Asian individuals and HLA-A30 for Africanindividuals.

Several suitable tumor-related peptides are described in Speiser andRomero (Seminars in Immunology 22 (2010) 144-154) and referencestherein. Most of them are HLA-A2 restricted, for example peptidesderived from Melan-A/MART-1, one of the gp100 epitopes and tyrosinasefor the melanocyte differentiation antigens; prostate surface antigenand PSAP for prostate; carcinoembryonic antigen and MUC-1 for mucosaltumors; HER-2/neu for breast carcinoma; G250 for renal cell carcinoma;the PR1 shared by two myeloid leukemia associated antigens, PR3 andneutrophil elastase which are normally expressed in granulocytes andoverexpressed in myeloid leukemia cells; the shared tumor-specificantigens MAGE-A and NY-ESO-1 for various tumor types; and theoverexpressed proteins survivin and telomerase. Suitable InfluenzaA-derived peptides are described in Wu et al (supra).

Therefore, in a further embodiment, the peptide derived from theantigen-comprising protein is a peptide presented by a MHC-I, preferablyby allele HLA-A2, HLA-A1, HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30,more preferably by allele HLA-A2. Methods for identifying such peptidesare described and summarized in Wu et al. (supra). For example, thesystematic identification approach of Wu et al. (supra) may be used, orsuitable algorithms described therein.

Therefore, in a further embodiment, the peptide derived from theantigen-comprising protein has a length of 8, 9 or 10 amino acids and isa peptide presented by a MHC-I, preferably by allele HLA-A2, HLA-A1,HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30, more preferably by alleleHLA-A2.

In a further preferred embodiment of the medicament for use, thereceptor on the surface of a dendritic cell is a receptor on the surfaceof cross-presenting dendritic cells.

Cross-presenting dendritic cells are particularly suitable forpresenting peptides in the context of MHC-I. A cross-presenting DC iscapable to take up soluble or targeted protein, process it, and presentit in the context of MHC-I. All conventional DC are capable of antigencross-presentation. Quantitatively optimal antigen cross-presentation isdone by XCR1⁺ DC in the mouse and by XCR1⁺ DC within the BDCA3⁺ DCpopulation in the human. Therefore, by XCR1⁺ DC are preferred murinecross-presenting DC, and XCR1⁺ DC within the BDCA3⁺ DC population arepreferred human cross-presenting DC.

In a further preferred embodiment of the medicament for use, thereceptor on the surface of a dendritic cell is chemokine (C motif)receptor 1 (XCR1), nectin-like molecule 2, a c-type lectin (CLEC) suchas CLEC9A.

C-type lectins are Ca⁺⁺-dependent glycan-binding proteins that shareprimary and secondary structural homology in theircarbohydrate-recognition domains (CRDs). These proteins have a C-typelectin fold, which is a fold with highly variable protein sequence thatis also present in many proteins that do not bind carbohydrates.

The sequence of the human receptor CLEC9A is for example described inCaminschi et al., 2008, Blood 112, 3264-3273. A suitable moleculebinding to CLEC9A is e.g. a specific monoclonal anti-CLEC9A antibody.

The human receptor nectin-like molecule 2 is described in Takai et al.,2003, Cancer Sci 94, 655-667. A suitable molecule binding to nectin-likemolecule 2 is e.g. a specific anti-nectin-like molecule 2 monoclonalantibody.

The delivery system is particularly suitable for influencing the Th1response, and optionally also the Th2 response, in the immune system.

XCR1 is a chemokine receptor and is so far the only member of the “C”sub-family of chemokine receptors. It is also known as GPR5 or CCXCR1.GPR5, cloned previously as an orphan G-protein coupled receptor, hasbeen recognized first in the human and then in the mouse as amonospecific receptor for XCL1 and was accordingly referred to as XCR1.

The natural ligand of XCR1 is XCL1, which is also known as ATAC,lymphotactin or SCM-1. It is the only member of the C family ofchemokines. Activation-induced, T cell-derived, and chemokine-relatedcytokine (ATAC) was cloned in the human (Muller et al., 1995, Eur. J.Immunol. 25, 1744-48), and independently as lymphotactin (Kelner et al.,1994, Science 266, 1395-99) in the mouse and SCM-1 (Yoshida et al.,1995, FEBS Lett. 360, 155-9) in the human. According to the nomenclatureon chemokines ATAC/lymphotactin/SCM-1 is now designated “XCL1”. XCL1 issecreted mainly by activated CD8⁺ T-cells, Th1 CD4⁺ T cells and by NKcells. In the human, a variant of XCL1 designated XCL2 has beendescribed in which the amino acids aspartate and lysine in position 28and 29 of the full length protein are exchanged for histidine andarginine, respectively (Yoshida et al., 1996, FEBS Lett. 395, 82-8),which may also be used for the present invention. An exemplary method toproduce XCL1 in biologically active form is described in Example 8 of WO2009/065561. Analogous methods may be used in order to produce otherbiologically active forms of XCL1, e.g. those of other species.

In an even more preferred embodiment, the receptor on the surface of adendritic cell is XCR1.

The amino acid sequence of human XCR1 is known (NCBI; accessionNP_001019815):

(SEQ ID NO: 12) MESSGNPEST TFFYYDLQSQ PCENQAWVFA TLATTVLYCLVFLLSLVGNS LVLWVLVKYE SLESLTNIFI LNLCLSDLVFACLLPVWISP YHWGWVLGDF LCKLLNMIFS ISLYSSIFFLTIMTIHRYLS VVSPLSTLRV PTLRCRVLVT MAVWVASILSSILDTIFHKV LSSGCDYSEL TWYLTSVYQH NLFFLLSLGIILFCYVEILR TLFRSRSKRR HRTVKLIFAI VVAYFLSWGPYNFTLFLQTL FRTQIIRSCE AKQQLEYALL ICRNLAFSHCCFNPVLYVFV GVKFRTHLKH VLRQFWFCRL QAPSPASIPH SPGAFAYEGA SFY

In a further preferred embodiment of the medicament for use, themolecule of a) is a ligand to the receptor or an antibody or antibodyfragment against the receptor. In an even more preferred embodiment, thereceptor is chemokine (C motif) receptor 1 (XCR1) and the molecule of a)is anti-XCR1 antibody or fragment thereof or chemokine (C motif) ligand1 (XCL1) or a functionally active variant thereof, particularlycomprising or consisting of the sequence of any of SEQ ID NOs: 7 to 10,preferably, of SEQ ID NOs: 8 to 10, more preferably of SEQ ID NOs: 9 or10, especially of SEQ ID NO: 10.

The amino acid sequences of XCL1 (ATAC) of several species (includinghuman: SEQ ID NO: 1, GenBank accession P47992; mouse: SEQ ID NO: 2,GenBank accession P47993; and rat SEQ ID NO: 3, GenBank accessionP51672) are known and are shown as SEQ ID NO: 1 to 3 (see below).Additionally, a specific XCLR1 agonist referred to as K4.1 HHV8 (SEQ IDNO: 4, GenBank accession AAB62672.1) (see below), which is a viralchemokine-like protein, is also known. Any of these naturally occurringXCR1 ligands or any other natural occurring XCR1 ligand may be used.

Alternatively, a functionally active variant of any naturally occurringXCL1 may be used. The term variant encompasses fragments, variantsderived by one or more amino acid additions, deletions and/orsubstitutions and molecules, particularly proteins, comprising anynaturally occurring XCL1 or part thereof, such as fusion proteins. TheXCL1 portion of the fusion protein may be flanked by the amino acidresidue(s) C-terminally, N-terminally, or C- and N-terminally.

The functionally active fragment is characterized by being derived fromany natural occurring XCR1 ligand, particularly XCL1, especially thoseof SEQ ID NO:1 to 4, by one or more amino acid deletions. Thedeletion(s) may be C-terminally, N-terminally and/or internally.Preferably, the fragment is obtained by at most 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50 or 60, more preferably by at most 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25 or 30, even more preferably at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, still more preferably atmost 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, most preferably 1, 2, 3, 4 or 5amino acid deletion(s). The functionally active fragment of theinvention is characterized by having a biological activity similar tothat displayed by the ligand from which it is derived, including theability to binding to XCR1 and mediate internalization of a protein of(b). The fragment of the naturally occurring XCR1 ligand, particularlyXCL1, especially those of SEQ ID NO:1 to 4, is functionally active inthe context of the present invention, if the activity (binding as wellas internalization) of the fragment amounts to at least 10%, preferablyat least 25%, more preferably at least 50%, even more preferably atleast 70%, still more preferably at least 80%, especially at least 90%,particularly at least 95%, most preferably at least 99% of the activityof the XCL1 without sequence alteration. These fragments may be designedor obtained in any desired length, including as small as about 18 to 50amino acids in length.

The functionally active fragment of the naturally occurring XCR1 ligand,particularly XCL1, especially those of SEQ ID NO:1 to 4, may be alsocharacterized by other structural features. Accordingly, in onepreferred embodiment of the invention the functionally active fragmentsconsists of at least 60%, preferably at least 70%, more preferably atleast 80%, still more preferably at least 90%, even more preferably atleast 95%, most preferably 99% of the amino acids of the XCR1 ligand ofany of the SEQ ID NOS: 1 to 4. The functional active fragment as definedabove may be derived from the peptide by one or more amino aciddeletions. The deletions may be C-terminally, N-terminally and/orinternally. The above sequence alignment of SEQ ID NOs: 1 to 4 showsdomains of the naturally occurring ligands which seem to be conserved.In a preferred embodiment of the invention, these domains should bemaintained in the fragment.

Conserved domains include those amino acids of the processed N-terminus(the processed N-terminus starting with amino acid 22 of non processedN-terminus) for SEQ ID NOs: 1 to 3 and with amino acid 27 for SEQ ID NO4) at positions 1-2 (V/S G), 13-27 (S/N L X T/S Q/A R L P V/P X K/R I/LK/I X T/G X Y, X=any or no amino acid; SEQ ID NO: 5), 35 to 51 (R/K A VI F I/V T K/H R/S G L/R K/R I/V C NG D/S P; SEQ ID NO: 6) and adisulfide bridge between cysteine residues at positions 11 and 48 (seealso above alignment). A consensus sequence for the sequences of SEQ IDNO: 1 to 4 isXGXXXXXXXXXXCXXXLXXXRLPXXXXXXXXYXXXXXXXXXXAVIFXTXXGXX-XCXXP (SEQ ID NO:7) if only identical amino acids are considered and(V/S)GX(E/A)(V/T)XXXXXXXC(V/E)X(S/N)LX(T/S)(Q/A)RLP(V/P)X(K/R)(I/L)(K/I)-X(T/G)XYX(I/T)X(E/T)(G/V)XXXX(R/K)AVIF(V/I)T(K/H)(R/S)G(L/R)(K/R)XC(A/G)-(D/S)P(SEQ ID NO: 8) if identical amino acids and majority amino acids (i.e.amino acids which are present in 3 of the 4 sequences, the alternativeamino acid is listed after the slash) are considered. A consensussequence for the sequences of SEQ ID NO: 1 to 3 isVGXEVXXXXXCVXLXTQRLPVXXIKTYXIXEGXXRA-VIFXTKRGLXXCADPXAXWVXXXXXXXDXXXXXXXXXXXTXPTXXQXSXXTAXT-LTG(SEQ ID NO: 9) if only identical amino acids are considered andVG(T/S)EV-(L/S)X(E/K)(S/R)XCV-(S/N)LXTQRLPV(Q/S)(K/R)IKTY(T/I)IXEG(A/S)(M/L)RAVIF(V/I)TKRGL(K/R)(I/V)-CADP(Q/E)A(K/T)WV(K/R)X(A/V)(I/V)(K/R)(T/S)(V/M)D(G/R)(R/K)(A/S)(S/N)(T/A)-(R/S)(K/N)(N/S)(M/K)(A/I)(E/Q)TXPT(G/Q)(A/T)Q(R/Q)S(T/A)(S/N)TA(V/I)TLTG(SEQ ID NO: 10) if identical amino acids and majority amino acids (i.e.amino acids which are present in 2 of the 3 sequences, the alternativeamino acid is listed after the slash) are considered.

Accordingly, in a preferred delivery system for use in the invention thefunctionally active variant, preferably the functionally activefragment, of XCL1 comprises or consists of the sequence of any of SEQ IDNOs: 7 to 10, preferably of SEQ ID NOs: 8 to 10, more preferably of SEQID NOs: 9 or 10, especially of SEQ ID NO: 10.

In a further preferred embodiment of the invention, a XCL1 variant asdefined above, wherein the XCR1 ligand is a functionally active variantof an XCR1 ligand of any of the SEQ ID NOS: 1 to 4 and wherein thevariant has at least 50% sequence identity to the XCR1 ligand of any ofthe SEQ ID NOS: 1 to 4 is used. In a more preferred embodiment thefunctionally active variant has a sequence identity of at least 60%,preferably at least 70%, more preferably at least 80%, still morepreferably at least 90%, even more preferably at least 95%, mostpreferably 99% to the antigen of any of the SEQ ID NOS: 1 to 4.

The percentage of sequence identity can be determined e.g. by sequencealignment. Methods of alignment of sequences for comparison are wellknown in the art. Various programs and alignment algorithms have beendescribed e.g. in Smith and Waterman, Adv. Appl. Math. 2: 482, 1981 orPearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444-2448, 1988.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215: 403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.Variants of an antigen of any of the sequences of SEQ ID NOS: 1 to 4 aretypically characterized using the NCBI Blast 2.0, gapped blastp set todefault parameters. For comparisons of amino acid sequences of at least35 amino acids, the Blast 2 sequences function is employed using thedefault BLOSUM62 matrix set to default parameters, (gap existence costof 11, and a per residue gap cost of 1). When aligning short peptides(fewer than around 35 amino acids), the alignment is performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Methods fordetermining sequence identity over such short windows such as 15 aminoacids or less are described at the website that is maintained by theNational Center for Biotechnology Information in Bethesda, Md.(http://www.ncbi.nlm.nih.gov/BLAST/).

Alternatively, the alignment of multiple sequences may be performedusing the MegAlign Sofware from DNAStar (Madison, Wis., USA) employingthe ClustalV alignment algorithm (Higgins et al., 1992, Comput. Appl.Biosci. 8, 189-91). In the above alignment this software was used andset to the following default parameters: gap penalty 10, gap lengthpenalty 10. Because of the very low homology, manual adjustments werenecessary for the inclusion of SEQ ID NO 4 into the alignment.

The functional active variant is obtained by sequence alterations in thenaturally occurring XCR1 ligand, wherein the XCR1 ligand with thesequence alterations retains a function of the unaltered XCR1 ligand,e.g. having a biological activity similar to that displayed by thenaturally occurring XCR1 ligand, including the ability to binding toXCR1 and mediate internalization of a protein of (b) in step i). Suchsequence alterations can include, but are not limited to, conservativesubstitutions, deletions, mutations and insertions. Thesecharacteristics of the functional active variant can be assessed e.g. asdetailed above.

In a still more preferred embodiment the functionally active variant ofXCL1 for use is derived from the naturally occurring XCR1 ligand of anyof the sequences of SEQ ID NOS: 1 to 4 by conservative substitutions.Conservative substitutions are those that take place within a family ofamino acids that are related in their side chains and chemicalproperties. Examples of such families are amino acids with basic sidechains, with acidic side chains, with non-polar aliphatic side chains,with non-polar aromatic side chains, with uncharged polar side chains,with small side chains, with large side chains etc. In one embodiment,one conservative substitution is included in the peptide. In anotherembodiment, two conservative substitutions or less are included in thepeptide. In a further embodiment, three conservative substitutions orless are included in the peptide.

Examples of conservative amino acid substitutions include, but are notlimited to, those listed below:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln; HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Asn Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

A suitable monoclonal anti-XCR1 antibody for use according to theinvention is for example mAb 6F8 disclosed in WO 2009/065561 or MARX10(Bachem et al., 2012, Front Immunol 3, 214).

An anti-XCR1 antibody or functionally active fragment thereof may beused in one preferred embodiment as molecule binding to XCR1 as receptorin step i). An anti-XCR1 antibody or functionally active fragmentthereof is capable of binding specifically to the XCR1. The functionallyactive fragment of the antibody is defined analogously to thefunctionally active fragment of XCL1 (see above), i.e. the functionallyactive fragment (a) is characterized by being derived from any anti-XCR1antibody by one or more amino acid deletions, such as C-terminal,N-terminal and/or internal deletions and (b) is characterized by havinga biological activity similar to that displayed by the anti-XCR1antibody from which it is derived, including the ability to binding toXCL1. Naturally occurring antibodies are proteins used by the immunesystem to identify and neutralize foreign objects. Each naturallyoccurring antibody has two large heavy chains and two small light chainsand can bind to a different antigen. The present invention includes, forexample, monoclonal and polyclonal antibodies, chimeric, single chain,and humanized antibodies, as well as Fab fragments, Fab, Fab′, F(ab′)2′,Fv, or the product of a Fab expression library. The antibody or antibodycomponent can further be modified to prolong its biological half-life orin other ways to make them more suitable for targeting. Antibodiesgenerated against XCR1 can be obtained by direct injection of XCR1 or afragment thereof into an animal or by administering XCR1 or a fragmentthereof to an animal, preferably a non-human. The antibody so obtainedwill then bind to XCR1. For preparation of monoclonal antibodies, anytechnique known in the art, which provides antibodies produced bycontinuous cell line cultures, e.g. a hybridoma cell line, can be used.The production of a suitable monoclonal antibody is also detailed in WO2009/065561. Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to XCR1. Also, transgenic mice or other organisms suchas other mammals may be used to express humanized antibodies to XCR1.

In a further preferred embodiment of the medicament for use,

-   -   the antigen-comprising protein of (b) is in a fusion protein        with the molecule of a); and/or    -   the antigen of the antigen-comprising protein of (b) is an        immunogen, a pathogen-derived antigen, or a tumor antigen.

Thus, in one preferred embodiment, the antigen-comprising protein of (b)is in a fusion protein with the molecule of a). Such fusion proteins canbe synthesized e.g. synthetically or recombinantly, preferablyrecombinantly. Such fusion proteins enable efficient targeting to thedendritic cells. For example, fusion proteins of antibody MARX10 withOVA and XCL1 with OVA were successfully used in the Examples.

An immunogen is an antigen that stimulates an immune response. Antigensare substances recognized by specific receptors on T cells (T-cellreceptor) and B cells (B-cell receptor) within the immune system and areusually proteins or polysaccharides. This includes parts (coats,capsules, cell walls, flagella, fimbrae, and toxins) of bacteria,viruses, and other microorganisms. In general, lipids and nucleic acidsare antigenic only when combined with proteins and polysaccharides.Non-microbial exogenous (non-self) antigens can include pollen, eggwhite, and proteins from transplanted tissues and organs or on thesurface of transfused blood cells.

Antigens can be categorized as endogenous or exogenous. A preferredantigen of the present invention is an exogenous antigen.

In case a pathogen-derived antigen is used, preferably of a virus,bacterium and/or eukaryotic parasite, the medicament is preferably foruse in inducing a cellular cytotoxic immune response to an infectionwith such pathogen.

In case a tumor antigen is used, the medicament is preferably for use ininducing a cellular cytotoxic immune response to such tumor.

Dendritic cells can present exogenous antigens via MHC class Imolecules, a process known as “cross-presentation”.

In a further preferred embodiment of the medicament for use, the firstadjuvant of c) and second adjuvant are independently an adjuvant whichsupports a Th-1-mediated response, preferably they are independentlyselected from the group consisting of synthetic or recombinantRIG-I-agonists, TLR ligands, like poly ICLC and resiquimod (R848),Montanides, like ISA51, ISA720, saponins like Quil-A, ISCOM, QS-21, AS02and AS01, polyinosinic:polycytidylic acid (poly I:C), alipopolysaccharide (LPS), and a CpG oligodeoxynucleotide, morepreferably selected from an RIG-I-agonist, and a TLR ligand, such asresiquimod (R848), poly ICLC and polyinosinic:polycytidylic acid (polyI:C).

If both a first adjuvant of c) and a second adjuvant are used, they maybe the same or they may be different from each other.

Poly-ICLC consists of poly-IC stabilized with poly-L-lysine andcarboxymethylcellulose and is potent in supporting a Th1 response.

R848 is a selective ligand for TLR7 in mice and for TLR7 and TLR8 inhumans and activates the NLR pryin domain containing 3 (NLRP3)inflammasome.

For Poly-ICLC and R848, it is referred to Tacken and Figdor (supra) andreferences cited therein.

Montanides like ISA51 and ISA720 are water-in-oil emulsions containingsqualene and mannide-monooleate as an emulsifier, as disclosed inSpeiser and Romero and references cited therein.

Saponins like Quil-A, ISCOM, QS-21, AS02 and AS01 are triterpeneglycosides isolated from plants, as disclosed in Speiser and Romero andreferences cited therein.

An adjuvant is an agent which modifies the effect of other agents whilehaving few if any direct effects when given by itself. In pharmacology,adjuvants are drugs that have few or no pharmacological effects bythemselves, but may increase the efficacy or potency of other drugs whengiven at the same time. In immunology an adjuvant is an agent which,while not having any specific antigenic effect in itself, may stimulatethe immune system, increasing the response to a vaccine. The adjuvantsused in the present invention are preferably supporting a Th1 response.An adjuvant supporting or inducing a Th1 response (or type 1 T cellresponse) is understood as adjuvant which is capable to induce IL-12 inDC, and leads to the secretion of IL-2, IFN-γ and TNF-α by responsiveantigen-reactive CD8+ and CD4+ T cells, as well as production of thecytotoxic molecules granzyme B and perforin by antigen-reactivecytotoxic CD8+ and CD4+ T cells. Suitable preferred first and secondadjuvants which support a Th1 response are known in the art and are forexample described in Tacken and Figdor, and in Speiser and Romero.(Tacken P. J., Figdor C. G.; Targeted antigen delivery and activation ofdendritic cells in vivo: Steps towards cost effective vaccines. Semin.Immunol. (2011), doi:10.1016/j.smim.2011.01.001; Speiser D. E. andRomero P., Seminars in Immunology 22 (2010) 144-154).

The dendritic cell (DC) is capable of sensing through a large set of“danger signal” recognition receptors (e.g. toll-like receptors,NOD-like receptors), whether the antigen is of dangerous nature orwhether it is harmless. The patterns recognized by the “danger signal”recognition receptors (also designated “pattern recognition receptors”)are usually molecular structures that are unique to microorganisms.These can be cell wall components (e.g. lipopolysaccharide,peptidoglycan) or nucleic acid modifications (e.g. unmethylated CpGmotifs) in case of microbes, or structural features and modificationsthat are unique to viral DNA or viral RNA (e.g. double-stranded RNA).Also cells dying from apoptosis in the body release molecules which arecapable of triggering “danger signal” recognition receptors (e.g. HighMobility Group Protein B1, heat-shock proteins). Such danger signals arepreferred first and second adjuvants of the present invention.

In a further preferred embodiment of the medicament for use, there-activator is a peptide-loaded major histocompatibility complex classI (MHC-I) presenting cell and a second adjuvant, wherein the cell is ablood cell, especially a peripheral blood mononucleated cell (PBMC),preferably in combination with IL-2cx.

In a further preferred embodiment of the medicament for use, the T cellis a CD8+ T cell or a CD4+ T cell, preferably a CD8+ T cell.

In particular, the re-activation of CD8+ T cells is specificallyadvantageous for obtaining a strong and enhanced cytotoxic immuneresponse.

In a further preferred embodiment of the medicament for use, the timeframe is from 72 h to 12 days, 72 h to 9 days, particularly from 5 daysto 8 days, 5 days to 9 days or 5 days to 12 days.

In one preferred embodiment, the method steps i) and ii) are performedonly once.

However, it is possible to repeat the method steps with the samedelivery system and the same reactivator when the immune system hassettled again. This is typically the case after at least one month,preferably after at least two months after inducing a cellular cytotoxicimmune response according to the invention as described above.

Therefore, in another preferred embodiment, the method steps with thesame delivery system according to step i) and the same reactivatoraccording to step ii) is repeated after at least 1, 2, or 3 months afterperforming the method steps of the invention.

Alternatively, the method steps may be repeated with a differentdelivery system according to step i), preferably wherein the differentdelivery system comprises a different antigen-comprising protein (b) andoptionally a different molecule binding to a receptor on the surface ofa dendritic cell (a). In such embodiment, it is not necessary to waituntil the immune system has settled again. Therefore, the method maytherefore be repeated with a different delivery system e.g. 7 or 14 daysafter performing the method steps of the invention for the first timewith a first delivery system. In such embodiment, the same or adifferent reactivator may be used in step ii).

In another embodiment, the present invention relates to a medicament foruse in a method of extending a cellular cytotoxic immune responseagainst an antigen-comprising protein, the method comprising the stepof:

-   i) administering to a patient having T cells activated against an    antigen a peptide-loaded major histocompatibility complex class I    (MHC-I) presenting cell and a second adjuvant, wherein the peptide    is derived from the antigen-comprising protein, thereby    re-activating the activated T cell, and optionally further    administering complexed interleukin 2 (IL-2cx),    wherein the re-activator of step i) is administered in a time frame    of from 0 h to 14 days after the T cells were activated against an    antigen.

“Cellular cytotoxic immune response” is understood as Th1-type humoraland cellular (cytotoxic) immune reaction that can be elicited to a givenantigen. This is in contrast to the response of classical vaccines whichmainly address the Th2 antigen presentation pathway and mainly lead tothe generation of Th2-type (neutralizing) antibodies and immunereactions.

An “Extended” cellular cytotoxic immune response is understood as acellular cytotoxic immune response which occurs for a longer timecompared to the cellular cytotoxic immune response obtained by adelivery system of step i) of a medicament for use of the presentinvention. For example, the response may be extended by 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

For this embodiment, the same preferred embodiments apply, whereapplicable, as for above described medicaments for use of the presentinvention.

In an embodiment, the cell and the second adjuvant are administered in atime frame of from 0 h to 14 days after the T cells were activatedagainst an antigen.

In one preferred embodiment, the cell and the second adjuvant areadministered only once in a time frame of from 0 h to 14 days after theT cells were activated against an antigen.

In a further preferred embodiment, the cell and the second adjuvant areadministered in a time frame of from 48 h to 14 days after the T cellswere activated against an antigen.

For example, the cell and the second adjuvant are administered 48 h, 3days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days,12 days, 13 days or 14 days after the T cells were activated against anantigen.

In a more preferred embodiment, the cell and the second adjuvant areadministered in a time frame of from 5 days to 9 days, 5 days to 12 daysor 3 days to 9 days, even more preferably of from 4 days to 8 days or 5days to 12 days, for example 4 days, 5 days, 6 days, 7 days, 8 daysafter the T cells were activated against an antigen.

In another preferred embodiment, the cell and the second adjuvant areadministered only once in a time frame of from 0 h to 14 days after theT cells were activated against an antigen.

In a further preferred embodiment, the cell and the second adjuvant areadministered only once in a time frame of from 48 h to 14 days after theT cells were activated against an antigen.

For example, the cell and the second adjuvant are administered only once48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days or 14 days after the T cells were activatedagainst an antigen.

In a further preferred embodiment, the cell and the second adjuvant areadministered only once in a time frame of from 5 days to 9 days, 5 daysto 12 days or 3 days to 9 days, even more preferably of from 4 days to 8days or 5 days to 12 days, for example 4 days, 5 days, 6 days, 7 days, 8days after the T cells were activated against an antigen.

In one preferred embodiment, a combination of complexed interleukin 2(IL-2cx), and a peptide-loaded major histocompatibility complex class I(MHC-I) presenting cell and a second adjuvant is administered. In a morepreferred embodiment, the complexed interleukin 2 (IL-2cx) may beadministered before, concomitantly or after administration of thepeptide-loaded major histocompatibility complex class I (MHC-I)presenting cell and a second adjuvant. Preferably, the peptide-loadedmajor histocompatibility complex class I (MHC-I) presenting cell and asecond adjuvant are only administered once, and the complexedinterleukin 2 (IL-2cx) is administered repeatedly in a time frame offrom 0 h to 14 days after the T cells were activated against an antigen.In such embodiment, complexed IL-2 may be administered both before,concomitantly and/or after administration of the peptide-loaded majorhistocompatibility complex class I (MHC-I) presenting cell and a secondadjuvant.

For of the combination, the same preferred embodiments foradministration of peptide-loaded major histocompatibility complex classI (MHC-I) presenting cell and a second adjuvant, apply as for theadministration of the cells only.

In one embodiment, in case of the combination, the cell and the secondadjuvant are administered only once in a time frame of from 0 h to 14days after the T cells were activated against an antigen.

In a preferred embodiment, in case of the combination, the cell and thesecond adjuvant are administered in a time frame of from 48 h to 14 daysafter the T cells were activated against an antigen.

For example, in case of the combination, the cell and the secondadjuvant are administered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days,8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after theT cells were activated against an antigen.

In a more preferred embodiment, in case of the combination, the cell andthe second adjuvant are administered in a time frame of from 3 days to 9days, even more preferably of from 4 days to 8 days, for example 4 days,5 days, 6 days, 7 days, 8 days after the T cells were activated againstan antigen.

In another preferred embodiment, in case of the combination, the celland the second adjuvant are administered only once in a time frame offrom 0 h to 14 days after the T cells were activated against an antigen.

For example, the cell and the second adjuvant are administered only once48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days or 14 days after the T cells were activatedagainst an antigen.

In a further preferred embodiment, in case of the combination, the celland the second adjuvant are administered only once in a time frame offrom 3 days to 9 days, even more preferably of from 4 days to 8 days,for example 4 days, 5 days, 6 days, 7 days, 8 days after the T cellswere activated against an antigen.

In case of the combination, complexed interleukin 2 (IL-2cx) ispreferably administered repeatedly after the T cells were activatedagainst an antigen in a time frame of from 0 h to 14 days after the Tcells were activated against an antigen. It is further possible, thatfurther administration of complexed IL-2 takes place after 14 days afterthe T cells were activated against an antigen; however, at least oneadministration is in a time frame of from 0 h to 14 days.

In a further preferred embodiment, in case of the combination, complexedinterleukin 2 (IL-2cx) is preferably administered in a time frame offrom 48 h to 9 days, even more preferably of from 3 days to 8 days, forexample 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the T cellswere activated against an antigen.

In a yet further preferred embodiment, in case of the combination,complexed interleukin 2 (IL-2cx) is preferably administered repeatedlyin a time frame of from 48 h to 9 days, even more preferably of from 3days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8days after the T cells were activated against an antigen. Thus,complexed interleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 times in a time frame of from 48 h to 9 days, evenmore preferably of from 3 days to 8 days, for example 3 days, 4 days, 5days, 6 days, 7 days, 8 days after the T cells were activated against anantigen. For example, complexed interleukin 2 (IL-2cx) may beadministered daily or every two days. For example, complexed interleukin2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7days and 8 days after the T cells were activated against an antigen. Inanother example, complexed interleukin 2 (IL-2cx) may be administered 48h, 4 days, 6 days, and 8 days after the T cells were activated againstan antigen.

It is further possible, that further administration of complexed IL-2takes place after the indicated time of, e.g. 14 days, preferably 9 or 8days, after the administration of the delivery system of step i);however, at least one administration is in the indicated time frame.

Therefore, in a further preferred embodiment, in case of thecombination,

-   (A) the cell and the second adjuvant are administered in a time    frame of from 0 h to 14 days, preferably 48 h to 14 days, more    preferably in a time frame of from 3 days to 9 days, even more    preferably of from 4 days to 8 days, for example 4 days, 5 days, 6    days, 7 days, 8 days after the T cells were activated against an    antigen, even more preferably only once in a time frame of from 48 h    to 14 days, more particularly only once in a time frame of from 3    days to 9 days, even more particularly only once in a time frame of    from 4 days to 8 days, for example 4 days, 5 days, 6 days, 7 days, 8    days after the T cells were activated against an antigen, and-   (B) complexed interleukin 2 (IL-2cx) is administered in a time frame    of from 0 h to 14 days, more preferably 48 h to 9 days, even more    preferably of from 3 days to 8 days, for example 3 days, 4 days, 5    days, 6 days, 7 days, 8 days after the T cells were activated    against an antigen, most preferably administered repeatedly in a    time frame of from 0 h to 14 days, more preferably 48 h to 9 days,    even more preferably of from 3 days to 8 days, for example 3 days, 4    days, 5 days, 6 days, 7 days, 8 days after the T cells were    activated against an antigen.

It is further possible in case of the combination, that furtheradministration of complexed IL-2 takes place after 14 days after the Tcells were activated against an antigen; however, at least oneadministration is in a time frame of from 0 h to 14 days.

In a yet further preferred embodiment, in case of the combination,complexed interleukin 2 (IL-2cx) is preferably administered repeatedlyin a time frame of from 48 h to 9 days, even more preferably of from 3days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8days after the T cells were activated against an antigen. Thus,complexed interleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 times in a time frame of from 48 h to 9 days, evenmore preferably of from 3 days to 8 days, for example 3 days, 4 days, 5days, 6 days, 7 days, 8 days after the T cells were activated against anantigen. For example, complexed interleukin 2 (IL-2cx) may beadministered daily or every two days. For example, complexed interleukin2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7days and 8 days after the T cells were activated against an antigen. Inanother example, complexed interleukin 2 (IL-2cx) may be administered 0h, 48 h, 4 days, 6 days, and 8 days after the T cells were activatedagainst an antigen.

It is further possible, that further administration of complexed IL-2takes place after the indicated time of, e.g. 14 days, preferably 9 or 8days, after the T cells were activated against an antigen; however, atleast one administration is in the indicated time frame.

In another preferred embodiment, in case of the combination, the celland the second adjuvant are administered only once in a time frame offrom 0 h to 14, preferably 48 h to 14 days after the T cells wereactivated against an antigen.

Although we tested the effect of ADAS after targeting of antigen intoXCR1⁺ DC, one can conclude from our results that any system leading to asignificant initial activation (“priming”) or re-activation of CD8⁺ Tcells and/or CD4⁺ T cells by T cell receptor triggering in vivo can beamplified with the ADAS approach. The T cell population could also beinitially activated in vitro and later adoptively transferred in vivo,so that ADAS could then be used to amplify the T cell effector responsein vivo.

Therefore, in one embodiment, T cells activated against an antigen canbe obtained by performing a method as described above in a medicamentfor use in step i) using a delivery system.

In another preferred embodiment, T cells activated against an antigencan be obtained in vitro. To this end, unselected T cells, preferablyobtained from the patient to be treated, are co-cultured with antigen orantigen-comprising protein and APC, and antigen-responsive T cells areselected, e.g. by an IFN-γ secretion assay, and expanded with growthfactors. Alternatively, antigen-specific T cells are sorted usingappropriate tetramers or analogues thereof, exposed to antigen in thepresence of APC and expanded with growth factors.

In a preferred embodiment of the medicament for use, the MHC-Ipresenting cell is a blood cell, especially a peripheral bloodmononucleated cell (PBMC).

The MHC-I presenting cell is preferably a cell obtained from the patientto be treated. Methods for obtaining such cells are known to the skilledperson. For example, blood may be retrieved and PBMC cells may then beisolated.

In a further preferred embodiment of the medicament for use, thecomplexed interleukin 2 (IL-2cx) is administered, preferablyadministered repeatedly, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 times, even more preferably wherein the complexedinterleukin 2 (IL-2cx) is administered every 1 or 2 days, and/or isadministered repeatedly during 5 days to 1 month, even more preferably 1to 2 weeks.

In a further preferred embodiment of the medicament for use, the peptidederived from the antigen-comprising protein has a length of 8, 9 or 10amino acids and/or is a peptide presented by a MHC-I, preferably byallele HLA-A2, HLA-A1, HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30,more preferably by allele HLA-A2.

In a further preferred embodiment of the medicament for use, the patientis a mammal, in particular a human.

In a further preferred embodiment of the medicament for use, the methodof inducing a cellular cytotoxic immune response is for prophylacticallytreating or treating a tumor and/or an infection.

The medicament for use can be provided to protect from infection(“prophylactically treating”). Alternatively, such a medicament could beused for therapeutic purposes. The infected individual, which may not beable to mount a sufficient Th1 immune response to the pathogen, could beadministered a medicament designed to induce and/or extend a cellularcytotoxic response, and would thus become capable of containing oreradicating the infection (“treating”). Examples would be malaria,tuberculosis, leishmania, prion diseases, orthomyxoviruses and inparticular influenza, hepatitis A, hepatitis B, chronic hepatitis C, HIVand other lentiviruses, cytomegalovirus, herpes viruses,papillomaviruses, bunyaviruses, caliciviruses, filoviruses, flavivirusesand in particular hepatitis C virus, papillomaviruses, paramyxoviruses,a variety of respiratory viruses and other viruses, or any otherinfection specified in the description.

The vaccine could also be used to protect healthy individuals fromdeveloping tumors with known antigenic components (e.g. melanoma,prostate carcinoma) (“prophylactically treating a tumor”).Alternatively, medicament for use could be used to cure patients whoalready have developed tumors. Examples of such tumors would be humanvirus-induced tumors, in particular papillomavirus-induced tumors,HCV-induced tumors, hepatitis B-virus induced tumors and others viruseswhich induce tumors upon chronic infection. Moreover, suitable tumors tobe treated are spontaneously arising solid tumors (e.g. melanoma,prostate cancer, breast cancer, adenocarcinoma of the gut, lung cancer)and leukemias.

A pathogen or infectious agent is a biological agent, especially aliving microorganism, which causes disease or illness to its host.Pathogen, according to this invention, means preferably a virus,bacterium and/or eukaryotic parasite. A pathogen-derived antigen is anantigen derived from a pathogen.

Cross-presentation of antigen is also of central importance for theeradication of tumors in the body. Tumor cells and tumor antigens haveto be taken up, processed, and presented by DC to elicit an anti-tumorimmune response. Since the elimination of most tumors requires aneffective cytotoxic Th1 T cell response, cross-presentation of tumorantigens is essential. Thus, for an effective anti-tumor response,cross-presenting DC play a pre-eminent role. As shown in Example 5, themedicaments for use of the present invention exhibit a surprisingbeneficial effect in a tumor model.

In one embodiment, the medicament for use comprises, preferably consistsof, (a) a molecule binding to a receptor on the surface of a dendriticcell, and (b) an antigen-comprising protein bound to molecule of (a).

In another embodiment, the medicament for use comprises, preferablyconsists of, (a) a molecule binding to a receptor on the surface of adendritic cell, (b) an antigen-comprising protein bound to molecule of(a) and (c) a first adjuvant.

In yet another embodiment, the medicament for use comprises, preferablyconsists of (A) (a) a molecule binding to a receptor on the surface of adendritic cell, (b) an antigen-comprising protein bound to molecule of(a) and (c) a first adjuvant and (B) (d) complexed interleukin 2(IL-2cx), (e) a peptide-loaded major histocompatibility complex class I(MHC-I) presenting cell and a second adjuvant, or (f) a combination of(d) and (e), wherein the peptide is derived from the antigen-comprisingprotein as defined in (A).

In another embodiment, the present invention relates to a kit-of-partscomprising, preferably consisting of, a delivery system as defined aboveand a re-activator as defined above.

In yet another embodiment, the present invention relates to akit-of-parts comprising, preferably consisting of (A) complexedinterleukin 2 (IL-2cx), and (B) a peptide-loaded majorhistocompatibility complex class I (MHC-I) presenting cell and a secondadjuvant.

In a yet further embodiment, the present invention relates to a methodof extending a cellular cytotoxic immune response against anantigen-comprising protein, the method comprising the step of:

-   ii) administering to a patient having T cells activated against an    antigen a peptide-loaded major histocompatibility complex class I    (MHC-I) presenting cell and a second adjuvant, wherein the peptide    is derived from the antigen-comprising protein, thereby    re-activating the activated T cell, and optionally further    administering complexed interleukin 2 (IL-2cx),    wherein the re-activator of step i) is administered in a time frame    of from 0 h to 14 days after the T cells were activated against an    antigen.

In a yet further embodiment, the present invention relates to method ofinducing a cellular cytotoxic immune response, the method comprising thesteps of:

-   i) administering to a patient a delivery system comprising (a) a    molecule binding to a receptor on the surface of a dendritic    cell, (b) an antigen-comprising protein bound to molecule of (a)    and (c) a first adjuvant, wherein upon binding of the molecule    of (a) to the receptor, the protein of (b) is internalized and    processed in the dendritic cell and the antigen comprised in the    protein is presented on the surface of the dendritic cell, thereby    activating a T cell in the patient; and-   ii) administering to the patient a re-activator selected from the    group consisting of (d) complexed interleukin 2 (IL-2cx), (e) a    peptide-loaded major histocompatibility complex class I (MHC-I)    presenting cell and a second adjuvant, and (f) a combination of (d)    and (e), wherein the peptide is derived from the antigen-comprising    protein as defined in step i), thereby re-activating the T cell    activated in step i),    wherein the re-activator of step ii) is administered in a time frame    of from 0 h to 14 days after the administration of the delivery    system of step i).

For the methods of the present invention and the kits-of-parts of thepresent invention, the same embodiments apply as for the medicaments foruse of the present invention.

FIGURES

FIG. 1 shows induction of cytotoxic activity after targeting of antigeninto XCR1⁺ DC

FIG. 2 shows protection from infection or from seeding of cancer cellsby the induced cytotoxic activity

FIG. 3 shows amplification of CD8⁺ T cell cytotoxicity obtained byinjection of syngeneic lymphocytes loaded with antigenic peptideSIINFEKL (SEQ ID NO: 11)

FIG. 4 shows highly synergistic amplification of cytotoxic CD8⁺ T cellsby co-application of peptide-loaded cells and complexed IL-2

FIG. 5 shows synergistic effects of antigen targeting and co-applicationof peptide-loaded cells and complexed IL-2 in the treatment ofestablished tumors

FIG. 6 shows that the low frequency of cytotoxic CD8⁺ T cells afterpriming using various modes of vaccination can be strongly amplifiedwith ADAS. (A) C57BL/6 animals were injected on day 0 with 200 μg ofsoluble, non-targeted ovalbumin (OVA), or with 5 μg of mAb MARX10-OVA,DEC-205-OVA, 33D1-OVA, MOPC21-OVA; in all cases, 10 μg poly I:C wereco-injected as adjuvant. On day 5, blood samples were taken and thefrequencies of SIINFEKL-specific CD8⁺ T cells determined by flowcytometry using a specific tetramer. (B) On day 5, the immune responseto the OVA-derived peptide SIINFEKL was amplified with the ADASprocedure (injection of 10×10⁶ syngeneic splenocytes loaded withSIINFEKL together with 50 μg poly I:C as adjuvant). On day 10, theanimals were sacrificed and the frequencies of SIINFEKL-specific CD8⁺ Tcells were determined in the spleen using the tetramer. (C) C57BL/6Batf3-KO animals were immunized as described in (A) and ADAS-treated, asdescribed in (B), and the frequencies of SIINFEKL-specific CD8⁺ T cellsdetermined in the spleen on day 10.

FIG. 7 (A) R9-SIINFEKL polypeptide does not externally bind to the MHC-Igroove: C57BL/6 splenocytes were incubated at a density of 5×10⁶cells/ml with SIINFEKL peptide at 1 μM for 2 h at 37° C., 5% CO₂ incomplete RPMI1640 culture medium, or at a density of 2×10⁶ cells/ml withR9-SIINFEKL polypeptide at 1 μM for 4 h. Cells were washed twice, andstained with anti-SIINFEKL-H2K^(b) mAb (clone 25-D1.16) to determine theefficiency of peptide loading to the MHC-I groove. In addition, cellswere co-stained with various lineage markers to identify differentsplenic cell populations and analyzed by flow cytometry. (B) R9-SIINFEKLcan be used to transport antigen into the cytoplasmic compartment ofprimary cells and thus allow loading of MHC-I with derived peptides:C57BL/6 splenocytes were incubated at a density of 2×10⁶ cells withR9-SIINFEKL polypeptide at various concentrations (1-30 μM) for 7 h asabove. Thereafter, the cells were washed twice, stained with mAb25-D1.16 to determine the efficiency of peptide loading to the MHC-Igroove, co-stained with lineage markers, and analyzed by flow cytometry.With 30 μM polypeptide a significant cell death was observed (notshown). (C) R9-SIINFEKL loaded primary cells can be used for ADAS:C57BL/6 mice were injected i.v. on day 0 with 10×10⁶ R9-SIINFEKL loaded(5 μM for 7 h) splenocytes, or for comparison with 10×10⁶SIINFEKL-loaded (2 μM for 2 h) splenocytes, or with 2 μg MARX10-OVA (allwith 10 μg poly I:C) for priming, and the frequency and cytotoxicpotential (granzyme B, KLRG1, not shown) of CD8⁺ T cells were determinedon day 5. Alternatively, C57BL/6 mice were primed with 2 μg MARX10-OVAand 10 μg poly I:C, and subjected to ADAS on day 5 by injection i.v.with SIINFEKL-loaded (2 μM for 2 h) splenocytes and 50 μg poly I:C(positive control). In parallel, C57BL/6 mice were primed i.v. with 2 μgMARX10-OVA, or 2 μg 33D1-OVA, or 2 μg 1 D3-OVA, or 200 μg untargeted OVA(all together with 10 μg poly I:C) on day 0, and subjected to ADAS byinjection i.v. of R9-SIINFEKL-loaded (5 μM for 7 h) syngeneicsplenocytes and 50 μg poly I:C. The ADAS-induced expansion ofSIINFEKL-specific CD8⁺ T cells was determined on day 10 by flowcytometry using a specific tetramer. All CD8⁺ T cells exhibited markersindicative of cytotoxicity (granzyme B, KLRG1, not shown).

FIG. 8 Administration of antigen can be dissociated from application ofadjuvant in the priming step: (A) C57BL/6 mice were injected i.v. on day0 with 2 μg MARX10-OVA together with 10 μg poly I:C as adjuvant, mixedin one solution. Alternatively, mice were injected on day −1 with 10 μgof poly I:C and on day 0 with 2 μg MARX10-OVA. Alternatively, mice wereinjected on day 0 with 2 μg MARX10-OVA and on day 1 with 10 μg poly I:C.In each experimental group, blood samples were taken on day 5 and thefrequencies of SIINFEKL-specific CD8⁺ T cells determined by flowcytometry using a specific tetramer. (B) The mice described in (A) weresubjected to the ADAS amplification procedure (i.v. injection ofsplenocytes externally loaded with SIINFEKL together with 50 μg of polyI:C) and the frequencies of SIINFEKL-specific CD8⁺ T cells in the spleenwere determined by flow cytometry. Administration of antigen can bedissociated from application of adjuvant in the ADAS procedure.

FIG. 9 Highly synergistic amplification of cytotoxic CD8⁺ T cells bycombining ADAS and administration of complexed IL-15: C57BL/6 animalswere primed on day 0 with 2 μg MARX10-OVA and 10 μg poly I:C andanalyzed for the total number of SIINFEKL-specific CD8⁺ T cells in thespleen on day 5 (prime) using a specific tetramer and flow cytometry.Some of the primed animals were on day 5 subjected to ADAS only(injection of 10×106 SIINFEKL-loaded splenocytes together with 50 μgpoly I:C), some received ADAS and were injected i.p. with IL-2cx (asdescribed in Examples 4 and 5) on days 6, 7, and 8, other mice receivedADAS on day 5 and were injected i.p. with complexed IL-15 (IL-15cx) onday 6, 7, and 8. In all ADAS-treated groups, the total number ofSIINFEKL-specific cytotoxic CD8⁺ T cells was determined on day 9. Thedose of IL-15cx for 1 mouse was generated by incubating 2 μg IL-15(Peprotech #210-15) with 9.3 μg sIL-15R-Fc (R&D, #551-MR-100) at 37° C.for 20 min, PBS was added to 500 μl and the solution injected i.p.

FIG. 10 ADAS can amplify resting memory CD8⁺ T cells in anantigen-specific manner: C57BL/6 mice were on day −1 adoptivelytransferred with 2,000 or 10,000 of OT-I T cells and primed on day 0with MARX10-OVA and 10 μg poly I:C. ADAS (injection of 10×10⁶SIINFEKL-loaded splenocytes together with 50 μg poly I:C) was performedin all animals on day 5, and animals were analyzed for the frequency of(A) OT-I T cells and (B) endogenous, Thy 1.1-negative SIIFEKL-specificCD8⁺ T cells on days 10 and 40. Another group of mice was subjected toADAS again on day 69, and analyzed for the frequency of (A) OT-I T cellsand (B) endogenous SIINFEKL-specific CD8⁺ T cells on day 74.

FIG. 11 In vivo amplification of in vitro-activated antigen-specificCD8⁺ T cells by ADAS: Splenocytes were isolated from OT-I mice andcultured in complete medium with SIINFEKL peptide at 1.4 nM for 3 days.Thereafter, cells were washed with PBS and 5×10⁵ OT-I T cells (asdetermined by flow cytometry) were adoptively transferred into naïveC57BL/6 mice and the animals were treated by ADAS at various time pointsafter transfer. Shown is the effect on the frequency of transferred CD8⁺T cells when ADAS was performed on day 5 after adoptive transfer. Theproportion of adoptively transferred CD8⁺ T cells of all CD8⁺ T cellswas determined in the blood on day 9 using flow cytometry. Thefrequencies in the blood correspond in these experiments to thefrequencies in the spleen of the animals.

EXAMPLES Example 1 Induction of Cytotoxic Activity after Targeting ofAntigen into XCR1⁺ DC (FIG. 1)

The model antigen OVA was recombinantly fused to the XCR1-specific mAbMARX10 (Bachem, A. et al. Front Immunol 3 (2012) 214) or recombinantlyfused to the chemokine ligand XCL1 (Hartung, E. et al. J Immunol 194(2015) 1069-1079), which specifically binds to the XCR1-receptor. WhenMARX10-OVA or XCL1-OVA were injected i.v. into naïve C57BL/6 mice at lowlevels, the antigen was targeted into XCR1⁺ DC. If this antigen primingoccurred in the presence of an adjuvant (3 μg LPS, CpG, or 10 μg polyI:C), substantial cytotoxic activity was induced (shown are data withpoly I:C as adjuvant). This cytotoxic activity was tested by injectingthe primed animals on day 6 i.v. with target cells (spleniclymphocytes), which were previously loaded with SIINFEKL (SEQ ID NO: 11)in vitro, an OVA-derived peptide. The target cells were labeled afterloading with the fluorophore CFSE to a high degree, while non-loadedcontrol splenic lymphocytes were labeled with CFSE to a low degree. Asshown in FIG. 1A, priming animals with OVA+adjuvant resulted incytotoxic activity which eliminated almost all SIINFEKL-loaded targetcells. FIG. 1B shows a dose-response curve obtained with various amountsof antigen targeted to XCR1⁺ DC. Identical results were obtained afterusing MARX10-SIINFEKL or XCL1-SIINFEKL for targeting of the immunogenicpeptide into XCR1⁺ DC.

Example 2 Protection from Infection or from Seeding of Cancer Cells bythe Induced Cytotoxic Activity (FIG. 2)

C57BL/6 mice were primed with MARX10-OVA (containing 2 μg of OVA) andadjuvant (10 μg of poly I:C) or were left untreated. Five days later,all mice were infected with 1×10⁶ CFU (=5×LD₅₀) of a L. monocytogenesstrain, into which the peptide sequence SIINFEKL (SEQ ID NO: 11) hasbeen engineered recombinantly (Foulds et al., 2002, J. Immunol. 168,1528-1532). While all untreated mice died within 3 to 4 days, theinduced level of cytotoxicity by antigenic priming fully protected allanimals from disease (FIG. 2A).

C57BL/6 mice were primed with MARX10-OVA or XCL1-OVA (each containing0.16 μg of OVA) and adjuvant (3 μg LPS) i.v., control animals wereinjected with PBS. Seven days later, all animals were injected with5×10⁵ EG.7 cells, an aggressive syngeneic tumor line engineered toexpress OVA (Moore, M. W. et al. Cell 54 (1988) 777-785). WhilePBS-treated animals all exhibited strong tumor growth after 14 days,none of the immunized animals had any tumor tissue at the site ofinjection or elsewhere, indicating that the induced level ofcytotoxicity protected the animals from tumor seeding (FIG. 2B).

Example 3 Amplification of CD8⁺ T Cell Cytotoxicity Obtained byInjection of Syngeneic Lymphocytes Loaded with Antigenic PeptideSIINFEKL (SEQ ID NO: 11) (FIG. 3)

C57BL/6 mice were primed with MARX10-OVA (containing 2 μg OVA) and anadjuvant (poly I, CpG, or LPS, shown are the data with 10 μg poly I:C)on days −20, −15, −10, −7, −5, or -3. On day 0, the primed animals wereinjected i.v. with 10×10⁶ splenocytes (FIG. 3A) which were loaded beforeinjection with SIINFEKL (SEQ ID NO: 11) in vitro (“Antigen-DependentAmplification System”, ADAS). Together with the peptide-loaded cells anadjuvant was injected (various amounts of LPS or poly I:C), shown aredata with 50 μg of poly I:C. On day 10 animals were sacrificed and thetotal number of SIINFEKL-specific CD8⁺ T cells was determined in thespleen using SIINFEKL-specific tetramers and flow cytometry (FIG. 3A).The results demonstrated that the system used for amplification ofantigen-specific CD8⁺ T cells was effective in a narrow timeframebetween days 5 and 9 after the initial antigenic stimulation (FIG. 3A).

C57BL/6 mice were primed with MARX10-OVA (containing 2 μg OVA) and anadjuvant (10 μg poly I:C). Five days later, 10×10⁶ splenic lymphocytes,or purified T cells, dendritic cells, or B cells which were loadedbefore injection with SIINFEKL (SEQ ID NO: 11) in vitro were injectedi.v. into the primed animals. Together with the peptide-loaded cells anadjuvant was injected (50 μg poly I:C). On day 10 after priming withMARX10-OVA, the animals were sacrificed and the total number ofSIINFEKL-specific CD8⁺ T cells was determined in the spleen usingSIINFEKL-specific tetramers and flow cytometry. The results showed thatthe amplification of the cytotoxic CD8⁺ T cell response can be achievedwith various cell populations expressing MHC-I on the cell surface. Theamplified cells expressed high levels of effector molecules (TNF-α,IFN-γ, granzyme B).

Example 4 Highly Synergistic Amplification of Cytotoxic CD8⁺ T Cells byCo-Application of Peptide-Loaded Cells and Complexed IL-2 (FIG. 4)

C57BL/6 mice were primed with MARX10-OVA (containing 2 μg OVA) and anadjuvant (10 μg poly I:C). One group of primed animals was injected ondays 1, 2, and 3 after priming with complexed IL-2 (IL-2cx) obtained byincubating IL-2 (2 μg) and the anti-IL-2 mAb JES6-5H4 (10 μg, (Sander,B. J Immunol Methods 166 (1993) 201-214) overnight at 4° C., andsacrificed on day 6 (“Primed+IL-2cx”). Another group of mice wasinjected on day 5 with SIINFEKL-loaded splenocytes (10×10⁶) and adjuvant(50 μg poly I:C) and sacrificed on day 10 (“Primed+ADAS”). Another groupof mice was injected on day 5 with SIINFEKL-loaded splenocytes (10×10⁶)and adjuvant (50 μg poly I:C), and with IL-2cx on days 6, 7, 8, and 9,and sacrificed on day 10 (“Primed+ADAS+IL-2cx”). At the end of eachexperiment, the total number of SIINFEKL-specific CD8⁺ T cells wasdetermined in the spleen using SIINFEKL-specific tetramers and flowcytometry. The experiment determined a highly synergistic effect of ADASand IL-2cx in the amplification of antigen-specific cytotoxic CD8⁺ Tcells.

Example 5 Synergistic Effects of Antigen Targeting and Co-Application ofPeptide-Loaded Cells and Complexed IL-2 in the Treatment of EstablishedTumors (FIG. 5)

C57BL/6 mice were injected s.c. with 5×10⁵ EG.7 cells, an aggressivesyngeneic tumor line engineered to express OVA (Moore, M. W. et al. Cell54 (1988) 777-785). On day 6 after tumor injection mice were primed withMARX10-OVA (containing 2 μg OVA) and adjuvant (10 μg poly I:C), onegroup was left untreated. Six days after priming (day 11), one group ofmice was injected with SIINFEKL-loaded splenocytes (10×10⁶) and adjuvant(50 μg poly I:C) (“Primed+ADAS”). Another primed group of mice wasinjected on days 12, 13, 14, 15, 16, 17, 18, 19, 21, and 23 with IL-2cxonly (“Primed+IL-2cx”). Another primed group of mice was injected on day11 with SIINFEKL-loaded splenocytes (10×10⁶) and adjuvant (50 μg polyI:C), and with IL-2cx on days 12, 13, 14, 15, 16, 17, 18, 19, 21, and 23(“Primed+ADAS+IL-2cx”). On day 25 all mice were sacrificed (some controlanimals had to be sacrificed earlier, because the tumor became >1 cm indiameter). All data represent the mean average size of tumors (in mm²)in each treatment group (n=6). The results demonstrate that priming ofthe tumor-injected mice alone with targeted OVA was not effective. Incontrast, either IL-2cx or ADAS alone were effective by stronglyreducing the tumor mass for approximately 7-10 days in primed mice, butcould not prevent the outgrowth of the tumor thereafter. When acombination of ADAS and IL-2cx was applied to primed animals, the tumormass was strongly reduced and the tumor was fully controlled until theend of the experiment (some mice had fully rejected the tumor tissue).

Example 6 Various Modes of Immunization, Result in a Low-Frequency ofPrimed Antigen-Specific CD8⁺ T Cells. These can be Generally StronglyExpanded and Differentiated to Killer CD8⁺ T Cells with the ADASProcedure

MAb MARX10 (Bachem et al., Front Immunol 3 (2012) 214, EP2641915A1)recognizes XCR1, the lineage marker for XCR1⁺ DC, Mab DEC-205 (NLDC-145,Kraal et al., 1986, obtained from Biolegend) recognizes the CD205molecule expressed on murine XCR1⁺ DC, mAb 33D1 (Nussenzweig et al.,PNAS 79 (1982) 161-165, obtained from ATCC) recognizes the DCIR2molecule on SIRPα⁺ DC, mAb 1D3 recognizes CD19 on B cells (Krop et al.Eur J Immunol 26 (1996) 238-242, obtained from ATCC), mAb MOPC-21(Potter et al., J Natl Cancer Inst 26 (1961) 1109-1137, obtained fromBiolegend) does not recognize any molecule in the mouse and is thereforeused as an IgG1 isotype control. XCL1 is the chemokine ligand for XCR1and can be used for targeting of antigen to XCR1⁺ DC in the mouse and inthe human (Hartung et al. J Immunol 194 (2015) 1069-1079.

The antigen-binding regions of the heavy and light chains of mAbDEC-205, 33D1, 1 D3, MOPC-21 were identified by mass spectrometry andgrafted onto the backbone of mAb DEC-205 by standard recombinanttechniques, as described previously for mAb MARX10 (Hartung et al., JImmunol 194 (2015) 1069-1079). This backbone has been modifiedpreviously to minimize binding to Fc-receptors. All constructs were thenmodified in such a way as to accommodate OVA as a C-terminal fusionprotein to each of the antibodies, as described previously for mAbMARX10 (Hartung et al., J Immunol 194 (2015) 1069-1079). XCL1-OVA wasgenerated as described previously (Hartung et al., J Immunol 194 (2015)1069-1079).

C57BL/6 animals were injected on day 0 with a high amount (200 μg) ofsoluble, non-targeted OVA, or with 5 μg of mAb MARX10-OVA, DEC-205-OVA,33D1-OVA, MOPC21-OVA; in all cases, 10 μg poly I:C were co-injected asan adjuvant. On day 5, blood samples were taken and the frequencies ofOVA-specific CD8⁺ T cells determined by flow cytometry using a H-2K^(b)tetramer loaded with SIINFEKL and capable to bind to SIINFEKL-specificCD8⁺ T cells. As shown in FIG. 6A, all modes of antigen application,either non-targeted, or targeted to XCR1⁺ DC, to SIRPα⁺ DC, or to Bcells induced an initial expansion of SIINFEKL-specific CD8⁺ T cells(“priming”), resulting in a frequency of approximately 2-3% of all CD8⁺T cells in the blood. On day 5, the immune response to the OVA-derivedpeptide SIINFEKL was amplified with the ADAS procedure (injection of10×10⁶ SIINFEKL-loaded syngeneic splenocytes together with 50 μg polyI:C). On day 10, the animals were sacrificed and the frequency ofSIINFEKL-specific CD8⁺ T cells determined in the spleen. In addition,several markers indicative of cytotoxicity were determined (KLRG1,perforin, granzyme B, data not shown). In all cases, the ADAS procedureamplified the initial frequency of SIINFEKL-specific CD8⁺ T cellsapproximately tenfold (FIG. 6B) and induced a phenotype indicative ofcytotoxic T cells (not shown).

In parallel, Batf3-KO animals on the C57BL/6 background (animals whichlack XCR1⁺ DC, Hildner et al. Science 322 (2008) 1097-1100) were primedwith the non-targeted or targeted OVA reagents, as above. On day 5 theBatf3-KO animals were also treated by the ADAS procedure, as above. Onday 10, all animal were sacrificed and the frequencies ofSIINFEKL-specific CD8⁺ T cells were determined in the spleen. Whilepriming, followed by ADAS gave high frequencies of SIINFEKL-specificCD8⁺ T cells in all C57BL/6 animals (FIG. 6B), no substantialSIINFEKL-specific response could be observed after ADAS in any of theBatf3-KO animals (FIG. 6C).

Several conclusions can be drawn from these experiments. Immunizationusing high levels of non-targeted protein, when applied together with aTh1 adjuvant, will result in an initial frequency of antigen-specificcytotoxic CD8⁺ T cells, as demonstrated by us and others previously(Hartung et al., J Immunol 194 (2015) 1069-1079). Targeting of antigeninto DC makes this primary immunization much more effective, since onlylow amounts of antigen are required to achieve the same effect (Hartunget al, 2015, Caminschi et al. Front Immunol 3 (2012) 13). Surprisingly,even targeting of antigen to B cells (in this case via CD19, a surfacemolecule specifically expressed on B cells) was similarly effective totargeting of antigen to DC. This effect can either be explained bytransfer of antigen from B cells to DC (Allan et al. Immunity 25 (2006)153-162), or by “unspecific” binding of the targeting anybody toFc-receptors on DC. The latter effect is most likely responsible for theefficiency of priming when using MOPC-21, an isotype control antibodywhich does not recognize any antigen in the mouse immune system. Theseresults are fully compatible with earlier results, in which targeting ofantigen to marginal metallophilic macrophages via the surface receptorSiglec-1 also led to the generation of low-frequency cytotoxic CD8⁺ Tcells, but not in Batf3-KO animals (Backer et al. Proc Natl Acad Sci USA107 (2010) 216-221). Our experiments with Baf3-KO animals are in linewith the general assumption that priming of naïve T cells has to occurby DC. In particular, these experiments indicate that XCR1⁺ DC arerequired for this initial CD8⁺ T cell priming. Thus, targeting ofantigen into XCR1⁺ DC promises to be the most effective way of targetingprotein antigens, nucleic acids coding for antigens, or viral systemscoding for antigens in order to achieve a good primary CD8⁺ T cellresponse (“priming”).

Together, these results indicate that targeting of antigen usingantibodies or targeting using receptor ligands (e.g. XCL1-OVA) toconventional DC, skin DC or other DC, such as monocyte-derived DC, pDC,to macrophages or other cells is far more efficient for induction of aninitial cytotoxic response compared to the application of non-targetedantigen. It can be anticipated that all kinds of priming with proteinantigens (for example, but no limited to, by employing liposomes,nanoparticles, and other systems as antigen carriers (Saroja et al. IntJ Pharm Investig 1 (2011) 64-74) will give similar results, as long asthe protein is applied in the context of a Th1 adjuvant. It is also welldocumented in the literature that a similarly low initial frequency ofcytotoxic CD8⁺ T cells can be induced by non-protein immunization, suchas, but not limited to, application or injection of DNA or DNA-basedvaccines, or RNA or RNA-based vaccines, into the body using a variety ofsystems, either targeted or non-targeted (Saroja et al. Int J PharmInvestig 1 (2011) 64-74, Koup et al. Cold Spring Harb Perspect Med 1(2011) a007252, Ulmer et al. Vaccine 30 (2012) 4414-4418, Kramps et al.Wiley Interdiscip Rev RNA 4 (2013) 737-749) Similar priming of CD8⁺ Tcells can be achieved with viral systems or attenuated viruses (Draperet al. Nat Rev Microbiol 8 (2010) 62-73). Injection or application ofRNA or DNA-based preparations or non-replicating viral systems orattenuated viruses does not necessarily require an additional Th1adjuvant, since these agents are self-adjuvanted; i.e. these agents alsorepresent Th1 adjuvants themselves.

It is clear that in essentially all ways of vaccine delivery into thebody, the initial CD8⁺ cytotoxic response will be relatively weak. Sucha weak response will in many cases be insufficient to prevent infection,to treat an infection, or to eradicate cancerous tissue. Therefore,there is a need for a system which can strongly amplify the initialpriming.

In our experiments, we demonstrate that in all cases in which theinitial CD8⁺ cytotoxic response (priming) is insufficient to clear theinfection or the tumor, it can be amplified using the ADAS procedure.

Also immunization of patients with tumor tissue or dead cells will leadto a priming of the CD8⁺ T cell compartment (de Gruijl et al. CancerImmunol Immunother 57 (2008) 1569-1577) and thus will make these cellssusceptible to the ADAS procedure.

Alternatively, primary CD8⁺ T cell activation can be achieved byisolating pre-existing tumor or pathogen-specific CD8⁺ T cells from apatient, activating and expanding them in vitro, and injecting them backinto the patient. These adoptively transferred (re-injected) CD8⁺ Tcells will be re-activated and further expanded and differentiated tocytotoxic T cells by the ADAS procedure.

The ADAS amplification can be in all cases be further augmentedsynergistically by subsequent injections of complexed IL-2 (Examples 4,5) or complexed IL-15 (see Example 9).

In our experiments we used poly I:C as adjuvant. Similar results will beachieved with all type of Th1 adjuvants, such as, but not limited to,poly I:C, RIG-I agonists, and TLR8 agonists.

Example 7 Delivery of Antigen into the Cytoplasmic Compartment ofPrimary Cells for ADAS Leads to an Effective Loading of MHC-I withAntigen-Derived Peptides

We have demonstrated that external loading of the MHC-I of a variety ofprimary cells (e.g. B cells, T cells, DC, splenocytes) withantigen-derived peptide (e.g. SIINFEKL) can be used for ADAS. We haveanticipated that the same procedure will also work with a variety ofmethods introducing whole protein into primary cells or expressing aprotein inside the cell, as described above.

To further illustrate this concept we have used a stretch of 9 arginineresidues as a cell-penetrating peptide (CPP, Milletti 2012, Bechara etal., 2013) to transport an antigen-comprising protein (polypeptide) intoprimary cells. The entire sequence of this 37 aa-polypeptide (termedR9-SIINFEKL) is RRRRRRRRRGYPYDVPDYALEQLESIINFEKLTEWTS (SEQ ID No. 13).

R9-SIINFNEKL needs intracellular processing by the proteasome before aderived antigenic peptide (SIINFEKL) is presented on the cell surface inthe context of MHC-I. Thus, the system can serve as a model forintroducing a whole protein into a primary cell which is then processedby the proteasome into fragments, some of which are then presented onthe cell surface in the context of MHC-I.

This polypeptide cannot bind directly, externally, into the groove ofthe MHC-I. To prove this point, we incubated splenocytes of C57BL/6 micewith the polypeptide R9-SIINFEKL at 1 μM for 4 h 37° C. in completemedium. In parallel, splenocytes were incubated with the peptideSIINFEKL, which can directly bind to MHC-I externally, at 1 μM for only2 h. After incubation, the cells were washed and analyzed by flowcytometry using mAb 25-D1.16, which recognizes SIINFEKL in the contextof MHC-I H2K^(b) (Porgador et al. Immunity 6 (1997) 715-726). Whileincubation of splenocytes with SIINFEKL gave a strong signal withmacrophages, B cells, T cells, pDC, and DC, as expected (since SIINFEKLexternally binds to MHC-I), no signal was obtained with the R9-SIINFEKLpolypeptide (FIG. 7A). This experiment directly demonstrated that theR9-polypeptide cannot directly fit into the MHC-I groove and serve asantigen.

In the next step, splenocytes were incubated for 7 h at increasingconcentrations (1-30 μM) of the R9-SIINFEKL polypeptide. Afterincubation and washing, the amount of SIINFEKL-loaded MHC-I wasdetermined by staining with mAb 25-D1.16. As shown in FIG. 7B, allexamined primary splenic cells exhibited a dose-dependent signal.Although not directly measured, it can be assumed that also the MHC-IIwas loaded in a similar manner. This experiment demonstrated that apolypeptide, once transported into a cytoplasmic compartment of aprimary cell by a CPP, will be processed and presented in the context ofthe MHC-I (and MHC-II).

From this experiment, it can be deduced that the same procedure willalso work with a whole protein, which has also to be processed beforebeing presented in the context of the MHC-I (and MHC-II). In fact, asimilar loading of the MHC-I with SIINFEKL has been demonstrated withwhole OVA, to which a stretch of 9 arginine residues has been fusedN-terminally fused using standard recombinant techniques. In thatexperiment, OVA was correctly processed and presented in the context ofMHC-I, as determined by staining with mAb 25-D1.16 (Mitsui et al. JInvest Dermatol 126 (2006) 1804-1812). Thus, our experiment, which is inline with the literature, demonstrates that the introduction ofnon-processed polypeptides or proteins into the cell using the CPPprinciple will effectively load the MHC-I (and MHC-II) of this cell.

From this experiment, one can deduce that any type of internal loadingof primary cells with proteins and unprocessed peptides will lead to itan effective MHC-I presentation of peptides derived from this material.

Such an internal loading of primary cells could similarly be achievedwith other methods introducing peptides or proteins into cells, forexample by, but not limited to, electroporation, or by introducingnucleic acids coding for a peptides and proteins by a variety of methodssuch as, but not limited to, transfection, lipofection, transduction,injection, ballistic injection, infection.

We then tested the biological potency of primary cells internally loadedusing R9-SIINFEKL. To this end, splenocytes from C57BL/6 mice wereincubated with R9-SIINFEKL at 5 μM for 7 hours, and washed. They werethen injected on day 0 i.v. into naïve C57BL/6 animals and were comparedto i.v. injection of SIINFEKL-loaded splenocytes, or to the i.v.injection of MARX10-OVA. All preparation were applied together with polyI:C as adjuvant. As can be seen in FIG. 7C, all methods of antigenicdelivery induced a low frequency of SIINFEKL-specific cytotoxic CD8⁺ Tcells, as assessed with a specific tetramer and by phenotypic analysis(expression of granzyme B, KLRG1), MARX10-OVA being the most efficientmethod of antigen delivery.

In the next step, the R9-SIINFEKL loaded splenocytes were assessed fortheir capacity in the ADAS procedure. In this experiment, priming byMARX10-OVA and ADAS with SIINFEKL-loaded splenocytes served as thepositive control and gave around 20% of SIINFEKL-specific cytotoxic CD8⁺T cells (FIG. 7C). R9-SIINFEKL-loaded splenocytes were similarlyeffective in the ADAS procedure after priming with MARX10-OVA, 33D1-OVA,1D3-OVA or high amounts of non-targeted OVA as SIIFEKL-loadedsplenocytes (compare also FIG. 7A). This experiment demonstrated thatany type of effective internal loading of primary cells with antigenwill be efficient for ADAS. This loading could be with unprocessedpolypeptides, whole proteins, or with nucleic acids coding for peptidesor proteins, or using infectious agents, or viral or bacterial vectorsystems recombinantly modified to encode a desired polypeptide orprotein.

Example 8 Application of Th1 Adjuvant can be Dissociated in Time fromApplication of Antigen Both in the Priming Step and in the ADASProcedure

It is currently generally assumed that antigen has to be appliedtogether with an antigen in order to achieve immunization of the host.Therefore, antigen is usually mixed with the adjuvant and appliedtogether. It is currently generally assumed that the adjuvant shouldideally be even physically linked to the antigen to achieve optimalresults. Therefore, it was very surprising for us to realize that adissociation of antigen administration from adjuvant delivery in thepriming and ADAS procedures leads to good and even better immuneresponses.

C57BL/6 mice were injected i.v. on day 0 with 2 μg MARX10-OVA togetherwith 10 μg Poly I:C as adjuvant, mixed in one solution. Alternatively,mice were injected on day −1 with 10 μg of poly I:C and on day 0 with 2μg MARX10-OVA. Alternatively, mice were injected on day 0 with 2 μgMARX10-OVA and on day 1 with 10 μg poly I:C. In each experimental group,blood samples were taken on day 5 and the frequency of SIINFEKL-specificCD8⁺ T cells determined by flow cytometry. As can be seen in FIG. 8A,joint administration of antigen and adjuvant on day 0 gave a primingfrequency of around 2% of all CD8⁺ T cells. In contrast, application ofadjuvant one day before antigen appeared ineffective. Very surprisingly,administration of antigen on day 0 and application of adjuvant on day 1was most effective, giving a frequency of around 4% of all CD8⁺ T cellson day 5.

When all experimental groups were treated by the ADAS procedure (i.v.injection of splenocytes externally loaded with SIINFEKL and 50 μg ofpoly I:C), the group with the joint application of antigen and adjuvanthad around 15% SIINFEKL-specific cytotoxic CD8⁺ T cells. Clearly thebest result was achieved with the group in which adjuvant was applied 1day after antigen (around 30% SIINFEKL-specific CD8⁺ T cells).Interestingly, even in the group in which poly I:C was applied 1 daybefore antigen, there was a low level of SIINFEKL-specific T cells(around 5%), indicating that a certain priming has been achieved even inthis group (which then became measurable through the amplificationachieved in the ADAS procedure).

These experiments clearly demonstrate that application of antigen andadjuvant can be dissociated in time in the priming procedure. In fact,the results indicate that application of adjuvant some time afteradministration of antigen is advantageous over a joint application ofthe components. Our results indicate that application of adjuvant evenseveral days after application of antigen will be effective. The exacttime frame cannot be determined in the human and has to be estimatedalso as 1-2, possibly up to 3 days.

Example 9 Highly Synergistic Amplification of Cytotoxic CD8⁺ T Cells byCombining ADAS and Administration of Complexed IL-15

We have demonstrated that injection of complexed IL-2 (IL-2cx=murineIL-2 complexed with an antibody blocking the binding of IL-2 to its highaffinity receptor CD25) on days 1, 2, and 3 after priming withMARX10-OVA (2 μg) and poly I:C (10 μg) on day 0, did not substantiallyraise the number of primed CD8⁺ T cells on day 6. However, when theanimals were primed with MARX10-OVA (2 μg) and poly I:C (10 μg) on day 0and subjected to ADAS (injection of 10×10⁶ splenocytes loaded withSIINFEKL together with 50 μg poly I:C) on day 5, injection of IL-2cx ondays 6, 7, 8, and 9 dramatically raised the number of SIINFEKL-specificCD8⁺ T cells in the spleen on day 10 (Example 4 and FIG. 4).

Without ADAS, injection of IL-2cx on days 6, 7, 8, and 9 did notsubstantially raise the number of SIINFEKL-specific CD8⁺ T cells in thespleen (not shown), but was effective in reducing a tumor burden.Obviously, the ADAS procedure reactivates the primed CD8⁺ T cells insuch a manner that they become highly sensitive to the action of IL-2cxand this results in a strong expansion of cytotoxic CD8⁺ T cells.However, the exact molecular mechanism leading to this highly heightenedsensitivity to IL-2cx remains undetermined.

C57BL/6 animals were primed on day 0 with 2 μg MARX10-OVA and 10 μg polyI:C and subjected to ADAS (injection of 10×10⁶ SIINFEKL-loadedsplenocytes together with 50 μg poly I:C) on day 5. As describedearlier, the ADAS procedure raised the level of SIINFEKL-specific CD8⁺ Tcells from around 200,000 on day 5 to around 2×10⁶ cells after ADAS onday 10 (FIG. 9). ADAS-treated animals were then injected on days 6, 7,and 8 with complexed IL-15 (IL-15cx), or for comparison with IL-2cx (asdescribed above). On day 9, the total number of SIINFEKL-specific CD8⁺ Tcells was determined in the spleen using flow cytometry. As can be seenin FIG. 9, repeated injection of IL-15cx was similarly effective to therepeated injection of IL-2cx in strongly amplifying the number ofSIINFEKL-specific CD8⁺ T cells after the ADAS procedure. IL-15cx couldbe injected, like IL-2cx, i.v., s.c., i.p., or into a tumor to achievethis effect. Injection of IL-15cx also increased the cellular levels ofgranzyme B and expression of KLRG1, indicating augmented cytotoxicity(not shown).

Surprisingly, this experiment thus revealed that IL-15cx could stronglyamplify the levels of ADAS-reactivated antigen-specific CD8⁺ T cells,similar to the action of IL-2cx. From this experiment it can be deducedthat in all cases in which injection of IL-2cx was beneficial for theaugmentation of the cytotoxic immune response (as described above) alsoIL-15cx will be effective.

Example 10 The ADAS Procedure can be Used to Re-Activate and ExpandMemory CD8⁺ T Cells in an Antigen-Specific Manner

With naïve animals, which were primed with 2 μg MARX10-OVA and 10 μgpoly I:C on day 0, we could demonstrate that ADAS is rather ineffectivefor an amplification of the response when performed on day 3, butbecomes effective thereafter, and continues to be effective to a certainextent at least until day 20 (Example 3 and FIG. 3), and is mosteffective between days 4 and 9 in mice.

In these experiments, ADAS was used to amplify freshly activated naïveantigen-specific CD8⁺ T cells. The fact that ADAS only optimally worksin a certain time window following primary activation of CD8⁺ T cellsindicated that these T cells must be in a particular activation stage inorder to be sensitive for the ADAS amplification.

We therefore wondered whether ADAS could also be performed on restingmemory CD8⁺ T cells, which have a clearly different activation statuscompared to freshly activated CD8⁺ T cells. To this end, C57BL/6 micewere on day −1 adoptively transferred with 2,000 or 10,000 of OT-I CD8⁺T cells, which bear a T cell receptor specific for SIINFEKL presented inthe context of H-2K^(b). These transferred OT-I T cells bore the geneticmarker Thy1.1 (CD90.1), which made it possible to discriminate them fromendogenous T cells (compare Dorner et al., 2009). On day 0, the animalswere primed with MARX10-OVA together with 10 μg poly I:C. ADAS(injection of 10×10⁶ SIINFEKL-loaded splenocytes together with 50 μgpoly I:C) was performed on day 5, resulting in a frequency of 30% ofOT-I T cells of all CD8⁺ T cells in the spleen on day 10 for both groupsof adoptively transferred animals (FIG. 10A). When animals from the sameexperimental groups were analyzed on day 40, the frequency of their OT-ICD8⁺ T cells had declined to 1-2%, as expected (FIG. 10A). On day 69,another ADAS amplification was performed (injection of 10×10⁶splenocytes loaded with SIINFEKL together with 50 μg poly I:C) and thefrequency of OT-I T cells was determined in the spleen 5 days later (day74). Through the ADAS amplification, the frequency of OT-I T cells againrose to 30-60% of all CD8⁺ T cells in the spleen.

In all animals adoptively transferred with OT-I T cells, we alsoanalyzed the response of endogenous CD8⁺ T cells, since these could beidentified as CD8⁺ T cells negative for the Thy 1.1 marker, but stainingwith the SIINFEKL-tetramer. As can be seen in FIG. 10B, the endogenous,SIINFEKL-reactive CD8⁺ T cells showed a behaviour similar to the OT-I Tcells. In particular, the ADAS procedure could highly amplify endogenousSIINFEKL-specific memory T cells and these T cells bore all phenotypicmarkers typical of cytotoxic CD8⁺ T cells (granzyme B positivity,KLRG1-expression, data not shown).

These experiments determined that the ADAS procedure can reactivate andmassively expand resting memory CD8⁺ T cells in an antigen-specificmanner, so that they again become CD8⁺ cytotoxic effectors. Since theADAS procedure generates a state of T cells in which they are responsiveto IL-2cx and IL-15cx, both cytokine preparations will further stronglyaugment the response of memory CD8⁺ T cells to ADAS.

In summary, ADAS can not only amplify freshly activated naïve CD8⁺ Tcells in an antigen-specific manner in a certain time window afteractivation, but also resting memory CD8⁺ T cells.

Example 11 ADAS is Also Effective with In Vitro Activated, AdoptivelyTransferred CD8+ T Cells

In adoptive T cell therapy, antigen-specific CD8⁺ T cells (e.g. T cellsdirected to CMV antigens or any other pathogenic antigens or T cellsdirected to defined tumor antigens) are enriched from the PBMC of apatient. This is done, for example, using IFN-γ secretion capture afterin vitro stimulation of the PBMC with an antigenic peptide or proteinfrom this pathogen (e.g. CMV) or tumor, combined with magnetic cellsorting. The enriched antigen-specific CD8⁺ T cells are then furtherstimulated by addition of whole antigen or, more often, peptide antigen,and then expanded in vitro. After expansion, the activated CD8⁺ T cellsare then re-injected into the patient in order to achieve the desiredtherapeutic effect (elimination of the pathogen or tumor). Currently,the re-injected CD8⁺ T cells have a short life span in vivo, theircapacity to secrete IFN-γ is limited, and their cytotoxic potential issuboptimal. We have found that this shortcoming of the adoptive T celltherapy can be very substantially improved using the ADAS procedure.

Splenocytes of OT-I mice were in vitro activated with the peptideSIINFEKL for 3 days, without adjuvant. This activation of CD8⁺ T cellscould, however, also be done in the presence of a Th1 adjuvant. Afterculture, the cells (at this time composed of 97% OT-I CD8⁺ T cells) weretransferred into syngeneic C57BL/6 mice on day 0. ADAS (injection of10×10⁶ SIIFEKL-loaded splenocytes and 50 μg poly I:C i.v.) was appliedon either day 0, 1, 2, 3, 4, 5, or 6. ADAS on days 1, 2 or 3 did nothave any significant effect on the frequency of the adoptivelytransferred CD8⁺ T cells in the host animals. Clear amplification of thetransferred CD8⁺ T cells could, however, be observed with ADAS appliedon day 4, and optimal amplification was seen with ADAS on day 5 (FIG.11). At the same time, the expression of the surface marker KLRG1,indicative of maturation of the transferred CD8⁺ T cells to effectorcells, rose from 30-40% with ADAS on day 1 to 80-90% on days 5 and 6.These data allow concluding that the ADAS procedure is capable tostrongly amplify a population of in vitro-activated and adoptivelytransferred antigen-specific CD8⁺ T cells. At the same time, ADASinduces a further differentiation of these CD8⁺ T cells to cytotoxiceffector cells. The optimal time point for ADAS may differ in the human,since experiments in the mouse do not allow a precise prediction in thehuman. Thus, the period for optimal ADAS effects on the adoptivelytransferred CD8⁺ T cells may be more extended for example until 12 or 14days. It can also be anticipated that ADAS is not only effective withinthe short time frame after adoptive transfer of from 0 h to 14 days, butalso when the adoptively transferred CD8⁺ T cells have returned to thememory state after a prolonged period of time.

1. A medicament for use in a method of inducing a cellular cytotoxicimmune response, the method comprising the steps of: i) administering toa patient a delivery system comprising (a) a molecule binding to areceptor on the surface of a dendritic cell, (b) an antigen-comprisingprotein bound to molecule of (a) and (c) a first adjuvant, wherein uponbinding of the molecule of (a) to the receptor, the protein of (b) isinternalized and processed in the dendritic cell and the antigencomprised in the protein is presented on the surface of the dendriticcell, thereby activating a T cell in the patient; and ii) administeringto the patient a re-activator selected from the group consisting of (d)complexed interleukin 2 (IL-2cx), (e) a peptide-loaded majorhistocompatibility complex class I (MHC-I) presenting cell and a secondadjuvant, and (f) a combination of (d) and (e), wherein the peptide isderived from the antigen-comprising protein as defined in step i),thereby re-activating the T cell activated in step i), wherein there-activator of step ii) is administered in a time frame of from 0 h to14 days after the administration of the delivery system of step i). 2.The medicament for use of claim 1, wherein (x) the complexed interleukin2 (IL-2cx) is administered repeatedly, in particular 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 14 times, even more preferably wherein thecomplexed interleukin 2 (IL-2cx) is administered every 1 or 2 days,and/or is administered repeatedly during 5 days to 1 month, even morepreferably 1 to 2 weeks, and/or (xx) the peptide derived from theantigen-comprising protein has a length of 8, 9 or 10 amino acids and/oris a peptide presented by a MHC-I, preferably by allele HLA-A2, HLA-A1,HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30, more preferably by alleleHLA-A2.
 3. The medicament for use of claim 1 or 2, wherein the receptoron the surface of a dendritic cell is a receptor on the surface ofcross-presenting dendritic cells.
 4. The medicament for use of claim 2or 3, wherein the receptor on the surface of a dendritic cell ischemokine (C motif) receptor 1 (XCR1), nectin-like molecule 2, a c-typelectin (CLEC) such as CLEC9A, preferably the receptor on the surface ofa dendritic cell is XCR1.
 5. The medicament for use of any of claims 1to 4, wherein the molecule of a) is a ligand to the receptor or anantibody or antibody fragment against the receptor, particularly whereinthe receptor is chemokine (C motif) receptor 1 (XCR1) and wherein themolecule of a) is anti-XCR1 antibody or fragment thereof or chemokine (Cmotif) ligand 1 (XCL1) or a functionally active variant thereof,particularly comprising or consisting of the sequence of any of SEQ IDNOs: 7 to 10, preferably, of SEQ ID NOs: 8 to 10, more preferably of SEQID NOs: 9 or 10, especially of SEQ ID NO:
 10. 6. The medicament for useof any of claims 1 to 5, wherein the antigen-comprising protein of (b)is in a fusion protein with the molecule of a); and/or the antigen ofantigen-comprising protein of (b) is an immunogen, a pathogen-derivedantigen, or a tumor antigen.
 7. The medicament for use of any of claims1 to 6, wherein the first adjuvant of c) and second adjuvant areindependently an adjuvant which supports a Th-1-mediated response,preferably they are independently selected from the group consisting ofsynthetic or recombinant RIG-I agonists, TLR ligands, such as resiquimod(R848), poly ICLC or polyinosinic:polycytidylic acid (poly I:C)Montanides, saponins, a lipopolysaccharide (LPS), and a CpGoligodeoxynucleotide, more preferably selected from an RIG-I-agonist,and a TLR ligand, such as resiquimod (R848), poly ICLC orpolyinosinic:polycytidylic acid (poly I:C).
 8. The medicament for use ofany of claims 1 to 7, wherein the re-activator is a peptide-loaded majorhistocompatibility complex class I (MHC-I) presenting cell and a secondadjuvant, wherein the cell is preferably a blood cell, especially aperipheral blood mononucleated cell (PBMC), more preferably incombination with IL-2cx, and/or the cell and the second adjuvant areadministered only once in a time frame of from 0 h to 14 days after theadministration of the delivery system of step i).
 9. The medicament foruse of any of claims 1 to 8, wherein the T cell is a CD8+ T cell or aCD4+ T cell, preferably a CD8+ T cell.
 10. The medicament for use of anyof claims 1 to 9, wherein the time frame is from 72 h to 12 days,preferably 72 h to 9 days, more preferably from 5 days to 8 days, 5 daysto 9 days or 5 days to 12 days.
 11. A medicament for use in a method ofextending a cellular cytotoxic immune response against anantigen-comprising protein, the method comprising the step of: i)administering to a patient having T cells activated against an antigen apeptide-loaded major histocompatibility complex class I (MHC-I)presenting cell and a second adjuvant, wherein the peptide is derivedfrom the antigen-comprising protein, thereby re-activating the activatedT cell, and optionally further administering complexed interleukin 2(IL-2cx), wherein the re-activator of step i) is administered in a timeframe of from 0 h to 14 days after the T cells were activated against anantigen, preferably wherein the re-activator of step i) is administeredonly once in a time frame of from 0 h to 14 days after the T cells wereactivated against an antigen.
 12. The medicament for use of claim 11,wherein (x) the MHC-I presenting cell is a blood cell, especially aperipheral blood mononucleated cell (PBMC), and/or (xi) the complexedinterleukin 2 (IL-2cx) is administered, preferably administeredrepeatedly, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or14 times, even more preferably wherein the complexed interleukin 2(IL-2cx) is administered every 1 or 2 days, and/or is administeredrepeatedly during 5 days to 1 month, even more preferably 1 to 2 weeks,and/or (xii) the peptide derived from the antigen-comprising protein hasa length of 8, 9 or 10 amino acids and/or is a peptide presented by aMHC-I, preferably by allele HLA-A2, HLA-A1, HLA-A3, HLA-B7, HLA-B35,HLA-A24, or HLA-A30, more preferably by allele HLA-A2.
 13. Themedicament for use of any of claims 1 to 12, wherein the patient is ahuman.
 14. The medicament for use of any of claims 1 to 13, wherein themethod of inducing a cellular cytotoxic immune response is forprophylactically treating or treating a tumor and/or an infection. 15.Kit-of-parts comprising a delivery system as defined in any of claims 1and 5 and a re-activator as defined in claim 1 or 8.